Antisense compounds targeting genes associated with fibronectin

ABSTRACT

The present invention provides compounds comprising oligonucleotides complementary to a fibronectin transcript. Certain such compounds are useful for hybridizing to a fibronectin transcript, including but not limited to a fibronectin transcript in a cell. In certain embodiments, such hybridization results in modulation of splicing of the fibronectin transcript. In certain embodiments, such compounds are used to treat one or more symptoms associated with fibrosis. In certain embodiments, such compounds are used to treat one or more symptoms associated with renal fibrosis.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0197USC2SEQ.txt, created Apr. 29, 2019, which is 264 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Fibronectin is a high-molecular weight glycoprotein of the extracellularmatrix that binds to membrane-spanning receptor integrin proteins.Fibronectin has been implicated in a number of fibrotic disorders,including renal fibrosis. Alternative splicing of fibronectin pre-mRNAleads to the creation of fibronectin mRNA having a different combinationof exons, which in turn leads to the creation of several isoforms offibronectin protein. In certain instances, alternative splicing of thefibronectin gene results in a fibronectin protein isoform containing theextra type III domain A (EDA). Fibronectin containing extra type IIIdomain A (EDA) is implicated in the formation of fibrosis. See, e.g.,Muro et al., An Essential Role for Fibronectin Extra Type III Domain Ain Pulmonary Fibrosis, American Journal of Respiratory and Critical CareMedicine, Vol. 177, 638 (2008).

Antisense compounds have been used to modulate target nucleic acids.Antisense compounds comprising a variety of chemical modifications andmotifs have been reported. In certain instances, such compounds areuseful as research tools, diagnostic reagents, and as therapeuticagents. In certain instances antisense compounds have been shown tomodulate protein expression by binding to a target messenger RNA (mRNA)encoding the protein. In certain instances, such binding of an antisensecompound to its target mRNA results in cleavage of the mRNA. Antisensecompounds that modulate processing of a pre-mRNA have also beenreported. Such antisense compounds alter splicing, interfere withpolyadenlyation or prevent formation of the 5′-cap of a pre-mRNA.

Certain antisense compounds have been described previously. See forexample U.S. Pat. No. 7,399,845 and published International PatentApplication No. WO 2008/049085, which are hereby incorporated byreference herein in their entirety.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides compoundscomprising oligonucleotides. In certain embodiments, sucholigonucleotides are complementary to a fibronectin transcript. Incertain such embodiments, oligonucleotides are complementary to a targetregion of the fibronectin transcript comprising the EDA exon. In certainsuch embodiments, oligonucleotides are complementary to a target regionof the fibronectin transcript comprising an intron adjacent to the EDAexon. In certain such embodiments, oligonucleotides are complementary toa target region of the fibronectin transcript comprising an intronadjacent to the EDA exon and downstream of the EDA exon. In certain suchembodiments, oligonucleotides are complementary to a target region ofthe fibronectin transcript comprising an intron adjacent to the EDA exonand upstream of the EDA exon. In certain embodiments, the fibronectintranscript comprises an exonic splice enhancer for the EDA exon. Incertain embodiments, the fibronectin transcript comprises an exonicsplice silencer for the EDA exon. In certain embodiments,oligonucleotides inhibit inclusion of the EDA exon. In certainembodiments, oligonucleotides promote skipping of the of the EDA exon.In certain such embodiments, fibronectin mRNA without EDA mRNA isincreased. In certain such embodiments, fibronectin protein without EDAis increased.

The present disclosure provides the following non-limiting numberedembodiments:

Embodiment 1

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a fibronectintranscript.

Embodiment 2

The compound of embodiment 1, wherein the complementary region of themodified oligonucleotide is 100% complementary to the target region.

Embodiment 3

The compound of embodiment 1 or 2, wherein the complementary region ofthe modified oligonucleotide comprises at least 10 contiguousnucleobases.

Embodiment 4

The compound of embodiment 1 or 2, wherein the complementary region ofthe modified oligonucleotide comprises at least 12 contiguousnucleobases.

Embodiment 5

The compound of embodiment 1 or 2, wherein the complementary region ofthe modified oligonucleotide comprises at least 15 contiguousnucleobases.

Embodiment 6

The compound of embodiment 1 or 2, wherein the complementary region ofthe modified oligonucleotide comprises at least 18 contiguousnucleobases.

Embodiment 7

The compound of embodiment 1 or 2, wherein the complementary region ofthe modified oligonucleotide comprises at least 20 contiguousnucleobases.

Embodiment 8

The compound of any of embodiments 1-5, wherein the nucleobase sequenceof the oligonucleotide is at least 80% complementary to an equal-lengthregion of the fibronectin transcript, as measured over the entire lengthof the oligonucleotide.

Embodiment 9

The compound of any of embodiments 1-5, wherein the nucleobase sequenceof the oligonucleotide is at least 90% complementary to an equal-lengthregion of the fibronectin transcript, as measured over the entire lengthof the oligonucleotide.

Embodiment 10

The compound of any of embodiments 1-5, wherein the nucleobase sequenceof the oligonucleotide is 100% complementary to an equal-length regionof the fibronectin transcript, as measured over the entire length of theoligonucleotide.

Embodiment 11

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55469 and nucleobase 55790 of SEQ ID NO.: 1.

Embodiment 12

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55469 and nucleobase 55511 of SEQ ID NO.: 1.

Embodiment 13

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55511 and nucleobase 55732 of SEQ ID NO.: 1.

Embodiment 14

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55732 and nucleobase 55790 of SEQ ID NO.: 1.

Embodiment 15

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55491 and nucleobase 55511 of SEQ ID NO.: 1.

Embodiment 16

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55490 and nucleobase 55510 of SEQ ID NO.: 1.

Embodiment 17

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55491 and nucleobase 55513 of SEQ ID NO.: 1.

Embodiment 18

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55536 and nucleobase 55555 of SEQ ID NO.: 1.

Embodiment 19

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55576 and nucleobase 55600 of SEQ ID NO.: 1.

Embodiment 20

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55604 and nucleobase 55623 of SEQ ID NO.: 1.

Embodiment 21

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55610 and nucleobase 55697 of SEQ ID NO.: 1.

Embodiment 22

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55701 and nucleobase 55737 of SEQ ID NO.: 1.

Embodiment 23

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55738 and nucleobase 55757 of SEQ ID NO.: 1.

Embodiment 24

The compound of any of embodiments 1-10, wherein the target region iswithin nucleobase 55753 and nucleobase 55781 of SEQ ID NO.: 1.

Embodiment 25

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 5.

Embodiment 26

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 9.

Embodiment 27

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 13.

Embodiment 28

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 14.

Embodiment 29

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 15.

Embodiment 30

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 18.

Embodiment 31

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 22.

Embodiment 32

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 66.

Embodiment 33

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 67.

Embodiment 34

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises any of SEQ ID NOs: 5 to 24.

Embodiment 35

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises any of SEQ ID NOs: 30 to 90.

Embodiment 36

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 413.

Embodiment 37

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises SEQ ID NO: 346.

Embodiment 38

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises any of SEQ ID NOs: 104 to 176.

Embodiment 39

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises any of SEQ ID NOs: 177 to 329.

Embodiment 40

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises any of SEQ ID NOs: 403 to 435.

Embodiment 41

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises any of SEQ ID NOs: 105, 87, 126, 133, 134,140, 141, 147, 149, 157, 159, 190, 223, 238, 244, 268, 285, 300, 302,303, 308, 319, 327, 381, 382, 339, 346, 348, 364, 365, 367, 368, 369,370, 268, 276, 280, 406, 407, 412, 413, and 324.

Embodiment 42

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide has a nucleobase sequence comprising CTTCTTCT.

Embodiment 43

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide has a nucleobase sequence comprising GTTCC.

Embodiment 44

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide has a nucleobase sequence comprising GTCCC.

Embodiment 45

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises a sugar motif described by Formula I asfollows:

[(A)-(B)₂-(A)]_(n)

wherein:each A is independently a bicyclic nucleoside;each B is independently a 2′-substituted nucleoside or a2′-deoxynucleoside; and n is an integer from 3-6.

Embodiment 46

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises a sugar motif described by Formula II asfollows:

(A)₂-[(B)₂-(A)]_(n)-(A)

wherein:each A is independently a bicyclic nucleoside;each B is independently a 2′-substituted nucleoside or a2′-deoxynucleoside; andn is an integer from 3-6.

Embodiment 47

The compound of any of embodiments 1-24, wherein the antisenseoligonucleotide comprises a sugar motif described by Formula III asfollows:

(A)₂-[(B)₂-(A)]_(n)

wherein:each A is independently a bicyclic nucleoside;each B is independently a 2′-substituted nucleoside or a2′-deoxynucleoside; andn is an integer from 3-6.

Embodiment 48

The compound of any of embodiments 45 to 47, wherein each A comprises abicyclic nucleoside selected from among LNA and cEt.

Embodiment 49

The compound of any of embodiments 45 to 47, wherein each A comprises acEt modification.

Embodiment 50

The compound of any of embodiments 45 to 47, wherein each A comprises anLNA modification.

Embodiment 51

The compound of any of embodiments 45 to 50, wherein each B comprises a2′-substituted nucleoside having a 2′-modification selected from among2′-OMe, 2′-F, and 2′-MOE.

Embodiment 52

The compound embodiment 51, wherein the 2′-modification is a 2′-MOEmodification.

Embodiment 53

The compound of any of embodiments 45 to 50, wherein each B comprises a2′-deoxynucleoside.

Embodiment 54

The compound of any of embodiments 1-44, wherein the modifiedoligonucleotide comprises at least one modified nucleoside.

Embodiment 55

The compound of embodiment 54, wherein at least one modified nucleosidecomprises a modified sugar moiety.

Embodiment 56

The compound of embodiment 55, wherein at least one modified sugarmoiety is a 2′-substituted sugar moiety.

Embodiment 57

The compound of embodiment 56, wherein the 2′-substitutent of at leastone 2′-substituted sugar moiety is selected from among: 2′-OMe, 2′-F,and 2′-MOE.

Embodiment 58

The compound of embodiment 57, wherein the 2′-substituent of at leastone 2′-substituted sugar moiety is a 2′-MOE.

Embodiment 59

The compound of any of embodiments 54-56, wherein at least one modifiedsugar moiety is a bicyclic sugar moiety.

Embodiment 60

The compound of embodiment 59, wherein at least one bicyclic sugarmoiety is LNA or cEt.

Embodiment 61

The compound of any of embodiments 54-60, wherein at least one sugarmoiety is a sugar surrogate.

Embodiment 62

The compound of embodiment 61, wherein at least one sugar surrogate is amorpholino.

Embodiment 63

The compound of embodiment 61, wherein at least one sugar surrogate is amodified morpholino.

Embodiment 64

The compound of any of embodiment 1-63, wherein the modifiedoligonucleotide comprises at least 5 modified nucleosides, eachindependently comprising a modified sugar moiety.

Embodiment 65

The compound of embodiment 64, wherein the modified oligonucleotidecomprises at least 10 modified nucleosides, each independentlycomprising a modified sugar moiety.

Embodiment 66

The compound of embodiment 64, wherein the modified oligonucleotidecomprises at least 15 modified nucleosides, each independentlycomprising a modified sugar moiety.

Embodiment 67

The compound of any of embodiments 1 to 44 or 54 to 66, wherein eachnucleoside of the modified oligonucleotide is a modified nucleoside,each independently comprising a modified sugar moiety Embodiment 68. Thecompound of any of embodiments 1-67, wherein the modifiedoligonucleotide comprises at least two modified nucleosides comprisingmodified sugar moieties that are the same as one another.

Embodiment 69

The compound of any of embodiments 1-68, wherein the modifiedoligonucleotide comprises at least two modified nucleosides comprisingmodified sugar moieties that are different from one another.

Embodiment 70

The compound of any of embodiments 1-69, wherein the modifiedoligonucleotide comprises a modified region of at least 5 contiguousmodified nucleosides.

Embodiment 71

The compound of any of embodiments 1 to 44 or 54 to 70, wherein themodified oligonucleotide comprises a modified region of at least 10contiguous modified nucleosides.

Embodiment 72

The compound of any of embodiments 1 to 44 or 54 to 70, wherein themodified oligonucleotide comprises a modified region of at least 15contiguous modified nucleosides.

Embodiment 73

The compound of any of embodiments 1 to 44 or 54 to 70, wherein themodified oligonucleotide comprises a modified region of at least 20contiguous modified nucleosides.

Embodiment 74

The compound of any of embodiments 70-73, wherein each modifiednucleoside of the modified region has a modified sugar moietyindependently selected from among: 2′-F, 2′-OMe, 2′-MOE, cEt, LNA,morpholino, and modified morpholino.

Embodiment 75

The compound of any of embodiments 70-73, wherein the modifiednucleosides of the modified region each comprise the same modificationas one another.

Embodiment 76

The compound of embodiment 75, wherein the modified nucleosides of themodified region each comprise the same 2′-substituted sugar moiety.

Embodiment 77

The compound of embodiment 75, wherein the 2′-substituted sugar moietyof the modified nucleosides of the region of modified nucleosides isselected from 2′-F, 2′-OMe, and 2′-MOE.

Embodiment 78

The compound of embodiment 77, wherein the 2′-substituted sugar moietyof the modified nucleosides of the region of modified nucleosides is2′-MOE.

Embodiment 79

The compound of embodiment 75, wherein the modified nucleosides of theregion of modified nucleosides each comprise the same bicyclic sugarmoiety.

Embodiment 80

The compound of embodiment 79, wherein the bicyclic sugar moiety of themodified nucleosides of the region of modified nucleosides is selectedfrom LNA and cEt.

Embodiment 81

The compound of embodiment 75, wherein the modified nucleosides of theregion of modified nucleosides each comprises a sugar surrogate.

Embodiment 82

The compound of embodiment 81, wherein the sugar surrogate of themodified nucleosides of the region of modified nucleosides is amorpholino.

Embodiment 83

The compound of embodiment 81, wherein the sugar surrogate of themodified nucleosides of the region of modified nucleosides is a modifiedmorpholino.

Embodiment 84

The compound of any of embodiments 1-83, wherein the modified nucleotidecomprises no more than 4 contiguous naturally occurring nucleosides.

Embodiment 85

The compound of any of embodiments 1 to 44 or 54 to 85, wherein eachnucleoside of the modified oligonucleotide is a modified nucleoside.

Embodiment 86

The compound of embodiment 85 wherein each modified nucleoside comprisesa modified sugar moiety.

Embodiment 87

The compound of embodiment 86, wherein the modified nucleosides of themodified oligonucleotide comprise the same modification as one another.

Embodiment 88

The compound of embodiment 87, wherein the modified nucleosides of themodified oligonucleotide each comprise the same 2′-substituted sugarmoiety.

Embodiment 89

The compound of embodiment 88, wherein the 2′-substituted sugar moietyof the modified oligonucleotide is selected from 2′-F, 2′-OMe, and2′-MOE.

Embodiment 90

The compound of embodiment 89, wherein the 2′-substituted sugar moietyof the modified oligonucleotide is 2′-MOE.

Embodiment 91

The compound of embodiment 87, wherein the modified nucleosides of themodified oligonucleotide each comprise the same bicyclic sugar moiety.

Embodiment 92

The compound of embodiment 91, wherein the bicyclic sugar moiety of themodified oligonucleotide is selected from LNA and cEt.

Embodiment 93

The compound of embodiment 87, wherein the modified nucleosides of themodified oligonucleotide each comprises a sugar surrogate.

Embodiment 94

The compound of embodiment 93, wherein the sugar surrogate of themodified oligonucleotide is a morpholino.

Embodiment 95

The compound of embodiment 93, wherein the sugar surrogate of themodified oligonucleotide is a modified morpholino.

Embodiment 96

The compound of any of embodiments 1-95, wherein the modifiedoligonucleotide comprises at least one modified internucleoside linkage.

Embodiment 97

The compound of embodiment 96, wherein each internucleoside linkage is amodified internucleoside linkage.

Embodiment 98

The compound of embodiment 96 or 97, comprising at least onephosphorothioate internucleoside linkage.

Embodiment 99

The compound of embodiment 77, wherein each internucleoside linkage is amodified internucleoside linkage and wherein each internucleosidelinkage comprises the same modification.

Embodiment 100

The compound of embodiment 99, wherein each internucleoside linkage is aphosphorothioate internucleoside linkage.

Embodiment 101

The compound of any of embodiments 1-100 comprising at least oneconjugate.

Embodiment 102

The compound of any of embodiments 1-101 consisting of the modifiedoligonucleotide.

Embodiment 103

The compound of any of embodiments 1-102, wherein the compound modulatessplicing of the fibronectin transcript.

Embodiment 104

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising any of the sequences as set forth in SEQ ID NOs. 5 to 25 or30 to 90.

Embodiment 105

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 5.

Embodiment 106

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 9.

Embodiment 107

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 13.

Embodiment 108

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 14.

Embodiment 109

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 15.

Embodiment 110

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 18.

Embodiment 111

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 22.

Embodiment 112

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 66.

Embodiment 113

The compound of any of embodiments 1-103, having a nucleobase sequencecomprising the sequence as set forth in SEQ ID NO: 67.

Embodiment 114

The compound of any of embodiments 1-103, wherein the antisenseoligonucleotide has a nucleobase sequence comprising CTTCTTCT.

Embodiment 115

The compound of any of embodiments 1-103, wherein the antisenseoligonucleotide has a nucleobase sequence comprising GTTCC.

Embodiment 116

The compound of any of embodiments 1-103, wherein the antisenseoligonucleotide has a nucleobase sequence comprising GTCCC.

Embodiment 117

A pharmaceutical composition comprising a compound according to any ofembodiments 1-116 and a pharmaceutically acceptable carrier or diluent.

Embodiment 118

The pharmaceutical composition of embodiment 117, wherein thepharmaceutically acceptable carrier or diluent is sterile saline.

Embodiment 119

A method of decreasing the amount of EDA+ fibronectin protein in a cell,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 120

A method of increasing the amount of EDA− fibronectin protein in a cell,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 121

A method of reducing fibrosis, comprising contacting the cell with acompound according to any of embodiments 1-117.

Embodiment 122

A method of reversing fibrosis, comprising contacting the cell with acompound according to any of embodiments 1-117.

Embodiment 123

A method of reducing changes in cell phenotype due to fibrosis,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 124

A method of reversing changes in cell phenotype due to fibrosis,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 125

The method of embodiments 123-124, wherein the change in cell phenotypedue to fibrosis is the modulation of cadherin expression.

Embodiment 126

The method of embodiments 123-124, wherein the change in cell phenotypedue to fibrosis is the induction of a Smooth Muscle Actin (αSMA).

Embodiment 127

The method of embodiments 123-124, wherein the change in cell phenotypedue to fibrosis is the alteration of cortical f-actin localization.

Embodiment 128

The method of embodiments 123-124, wherein the change in cell phenotypedue to fibrosis is the induction of connexin 43 (Cx 43) expression.

Embodiment 129

The method of embodiments 123-124, wherein the change in cell phenotypedue to fibrosis is the increased secretion of MMP2 & MMP9.

Embodiment 130

The method of embodiments 123-124, wherein the change in cell phenotypedue to fibrosis is the alteration of the amount vimentin or thearrangement of vimentin within a cell.

Embodiment 131

The method of embodiments 123-124, wherein the change in cell phenotypedue to fibrosis is the alteration of the amount tight junction proteinZO-1 or the arrangement of tight junction protein ZO-1 within a cell.

Embodiment 132

A method of reducing loss of cell phenotype due to fibrosis, comprisingcontacting the cell with a compound according to any of embodiments1-117.

Embodiment 133

A method of reversing the loss of cell phenotype due to fibrosis,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 134

A method of increasing the ratio of EDA+/EDA− fibronectin in a cell,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 135

A method of decreasing the ratio of EDA+/EDA− fibronectin in a cell,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 136

A method of increasing the ratio of EDA−/EDA+ fibronectin in a cell,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 137

A method of decreasing the ratio of EDA−/EDA+ fibronectin in a cell,comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 138

A method of increasing the ratio of EDA+/EDA− fibronectin protein in acell, comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 139

A method of decreasing the ratio of EDA+/EDA− fibronectin protein in acell, comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 140

A method of increasing the ratio of EDA−/EDA+ fibronectin protein in acell, comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 141

A method of decreasing the ratio of EDA−/EDA+ fibronectin protein in acell, comprising contacting the cell with a compound according to any ofembodiments 1-117.

Embodiment 142

The method of any of embodiments 119-142, wherein the cell is in vitro.

Embodiment 143

The method of embodiments 119-142, wherein the cell is in an animal.

Embodiment 144

The method of embodiments 119-142, wherein the animal is a mouse.

Embodiment 145

The method of embodiments 119-142, wherein the animal is a human.

Embodiment 146

The method of any of embodiments 119-145, wherein TGFβ1 is present inthe cell.

Embodiment 147

The method of any of embodiments 119-146, wherein the healing and/orrestoration functions of the cell are not substantially affected.

Embodiment 148

A pharmaceutical composition comprising a compound according to any ofembodiments 1-117 and a pharmaceutically acceptable carrier or diluent.

Embodiment 149

The pharmaceutical composition of embodiment 148, wherein thepharmaceutically acceptable carrier or diluent is sterile saline.

Embodiment 150

A method comprising administering the pharmaceutical composition ofembodiments 148 or 149 to an animal.

Embodiment 151

The method of embodiment 150, wherein the animal is a mouse.

Embodiment 152

The method of embodiment 150, wherein the animal is a human.

Embodiment 153

The method of embodiment 150, wherein the administration is byinjection.

Embodiment 154

The method of embodiment 150, wherein the administration is systemic.

Embodiment 155

The method of embodiment 150 wherein the administration is local.

Embodiment 156

The method of any of embodiments 150-155, wherein the animal has one ormore symptom associated with fibrosis.

Embodiment 157

The method of embodiment 156, wherein the administration results inamelioration of at least one symptom associated with fibrosis.

Embodiment 158

The method of embodiment 156-157, wherein the fibrosis is renalfibrosis.

Embodiment 159

The method of embodiment 156-157, wherein the fibrosis is lung fibrosis.

Embodiment 160

The method of embodiment 156-157, wherein the fibrosis is liverfibrosis.

Embodiment 161

The method of embodiment 156-157, wherein the fibrosis is brainfibrosis.

Embodiment 162

The method of embodiment 156-157, wherein the fibrosis is muscularfibrosis.

Embodiment 163

The method of embodiment 156-157, wherein the fibrosis is cardiovascularfibrosis.

Embodiment 164

The method of embodiment 156-157, wherein the fibrosis is in the bone orthe bone marrow.

Embodiment 165

The method of embodiment 156-157, wherein the fibrosis is intestinalfibrosis.

Embodiment 166

The method of embodiment 156-157, wherein the fibrosis is epiduralfibrosis.

Embodiment 167

The method of any of embodiments 156-167, wherein the animal is a mouse.

Embodiment 168

The method of any of embodiments 156-167, wherein the animal is a human.

Embodiment 169

Use of the compound of any of embodiments 1 to 117 or the composition ofembodiments 148-149 for the preparation of a medicament for use in thetreatment of of at least one symptom associated with fibrosis.

Embodiment 170

Use of the compound of any of embodiments 1 to 117 or the composition ofembodiments 148-149 for the preparation of a medicament for use in theamelioration of one or more symptoms associated with fibrosis.

Embodiment 171

The use of any of embodiment 169-170, wherein the fibrosis is selectedfrom among renal, lung, liver, brain, muscular, cardiovascular, bone orbone marrow, intestinal, and/or epidural fibrosis.

Embodiment 172

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a target nucleicacid, wherein the modified oligonucleotide comprises a sugar motifdescribed by Formula I as follows:

[(A)-(B)₂-(A)]_(n)

wherein:each A is independently a bicyclic nucleoside;each B is independently a 2′-substituted nucleoside or a2′-deoxynucleoside; andn is an integer from 3-6.

Embodiment 173

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a target nucleicacid, wherein the modified oligonucleotide comprises a sugar motifdescribed by Formula II as follows:

(A)₂-[(B)₂-(A)]_(n)-(A)

wherein:each A is independently a bicyclic nucleoside;each B is independently a 2′-substituted nucleoside or a2′-deoxynucleoside; andn is an integer from 3-6.

Embodiment 174

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a target nucleicacid, wherein the modified oligonucleotide comprises a sugar motifdescribed by Formula III as follows:

(A)₂-[(B)₂-(A)]_(n)

wherein:each A is independently a bicyclic nucleoside;each B is independently a 2′-substituted nucleoside or a2′-deoxynucleoside; and n is an integer from 3-6.

Embodiment 175

The compound of any of embodiments 172 to 174, wherein each A comprisesa bicyclic nucleoside selected from among LNA and cEt.

Embodiment 176

The compound of any of embodiments 172 to 174, wherein each A comprisesa cEt modification.

Embodiment 177

The compound of any of embodiments 172 to 174, wherein each A comprisesan LNA modification.

Embodiment 178

The compound of any of embodiments 172 to 177, wherein each B comprisesa 2′-substituted nucleoside having a 2′-modification selected from among2′-OMe, 2′-F, and 2′-MOE.

Embodiment 179

The compound embodiments 178, wherein the 2′-modification is a 2′-MOEmodification.

Embodiment 180

The compound of any of embodiments 172 to 179, wherein each B comprisesa 2′-deoxynucleoside.

Embodiment 181

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a target nucleicacid, wherein the modified oligonucleotide comprises a kd₂kd₂kd₂kd₂kd₂kmotif, wherein each k comprises a cEt modification and each d comprisesa 2′-deoxynucleoside.

Embodiment 182

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a target nucleicacid, wherein the modified oligonucleotide comprises akkddkddkddkddkddkk motif, wherein each k comprises a cEt modificationand each d comprises a 2′-deoxynucleoside.

Embodiment 183

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a target nucleicacid, wherein the modified oligonucleotide comprises akkeekeekeekeekeeke motif, wherein each k comprises a cEt modificationand each e comprises a 2′-MOE modification.

Embodiment 184

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a target nucleicacid, wherein the modified oligonucleotide comprises a kddkddkddkddkddkmotif wherein each k comprises a cEt modification and each d comprises a2′-deoxynucleoside.

Embodiment 185

A compound comprising a modified oligonucleotide consisting of 8 to 30linked nucleosides and having a nucleobase sequence comprising acomplementary region comprising at least 8 contiguous nucleobasescomplementary to a target region of equal length of a target nucleicacid, wherein the modified oligonucleotide comprises a keekeekeekeekeekmotif, wherein each k comprises a cEt modification and each e comprisesa 2′-MOE modification.

In certain embodiments, including, but not limited to any of the abovenumbered embodiments, the fibronectin transcript is in a human. Incertain embodiments, including, but not limited to any of the abovenumbered embodiments, the fibronectin transcript is in a mouse.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of fibronectin splicing. Exons are representedas boxes and introns are represented as lines. Diagonal lines indicatesplicing pathways. As illustrated by the schematic, alternative splicingproduces two different mRNA products. Inclusion of the EDA exon resultsin mRNA containing the EDA exon (EDA+) which results in fibronectinprotein having EDA. Alternatively, exclusion of the EDA exon results inmRNA without the EDA exon (EDA−) and results in fibronectin proteinwithout EDA.

DETAILED DESCRIPTION OF THE INVENTION

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Inreference to an oligonucleotide, chemical modification does not includedifferences only in nucleobase sequence. Chemical modifications ofoligonucleotides include nucleoside modifications (including sugarmoiety modifications and nucleobase modifications) and internucleosidelinkage modifications.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugarmoiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl comprisingat least one substituent group that differs from that of a naturallyoccurring sugar moiety. Substituted sugar moieties include, but are notlimited to furanosyls comprising substituents at the 2′-position, the3′-position, the 5′-position and/or the 4′-position.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside is capable of (1) incorporation into an oligonucleotide and(2) hybridization to a complementary nucleoside. Such structures includerings comprising a different number of atoms than furanosyl (e.g., 4, 6,or 7-membered rings); replacement of the oxygen of a furanosyl with anon-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change inthe number of atoms and a replacement of the oxygen. Such structures mayalso comprise substitutions corresponding to those described forsubstituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugarsurrogates optionally comprising additional substituents). Sugarsurrogates also include more complex sugar replacements (e.g., thenon-ring systems of peptide nucleic acid). Sugar surrogates includewithout limitation morpholino, modified morpholinos, cyclohexenyls andcyclohexitols.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified.

As used herein, “heterocyclic base” or “heterocyclic nucleobase” means anucleobase comprising a heterocyclic structure.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” means an atom or group of atoms bound to anoligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid.

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measureable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenlyation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound hybridizes.

As used herein, “mRNA” means an RNA molecule that encodes a protein.

As used herein, “pre-mRNA” means an RNA transcript that has not beenfully processed into mRNA. Pre-RNA includes one or more intron.

As used herein, “transcript” means an RNA molecule transcribed from DNA.Transcripts include, but are not limited to mRNA, pre-mRNA, andpartially processed RNA.

As used herein, “fibronectin transcript” means a transcript transcribedfrom a fibronectin gene. In certain embodiments, a fibronectintranscript comprises SEQ ID NO: 1: the complement of GENBANK AccessionNo. NT_005403.14 truncated from nucleotides 66434501 to 66510708.

As used herein, “fibronectin gene” means a gene that encodes afibronectin protein and any fibronectin protein isoforms. In certainembodiments, a fibronectin gene is represented by GENBANK Accession No.NT_005403.14 truncated from nucleotides 66434501 to 66510708, or avariant thereof. In certain embodiments, a fibronectin gene is at least95% identical to GENBANK Accession No. NT_005403.14 truncated fromnucleotides 66434501 to 66510708. In certain embodiments, a fibronectingene is at least 90% identical to GENBANK Accession No. NT_005403.14truncated from nucleotides 66434501 to 66510708.

As used herein, “EDA− fibronectin protein” means a fibronectin proteinisoform that does not contain extra type III domain A.

As used herein, “EDA+ fibronectin protein” means a fibronectin proteinisoform that contains extra type III domain A.

As used herein, “EDA− fibronectin mRNA” means a fibronectin transcriptthat does not contain the extra type III domain A exon.

As used herein, “EDA+ fibronectin mRNA” means a fibronectin transcriptthat contains the extra type III domain A exon.

As used herein, “targeting” or “targeted to” means the association of anantisense compound to a particular target nucleic acid molecule or aparticular region of a target nucleic acid molecule. An antisensecompound targets a target nucleic acid if it is sufficientlycomplementary to the target nucleic acid to allow hybridization underphysiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity under stringent conditions. Complementary oligomericcompounds need not have nucleobase complementarity at each nucleoside.Rather, some mismatches are tolerated. In certain embodiments,complementary oligomeric compounds or regions are complementary at 70%of the nucleobases (70% complementary). In certain embodiments,complementary oligomeric compounds or regions are 80% complementary. Incertain embodiments, complementary oligomeric compounds or regions are90% complementary. In certain embodiments, complementary oligomericcompounds or regions are 95% complementary. In certain embodiments,complementary oligomeric compounds or regions are 100% complementary.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an antisense oligonucleotide specifically hybridizes tomore than one target site.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “motif” means a pattern of chemical modifications in anoligomeric compound or a region thereof. Motifs may be defined bymodifications at certain nucleosides and/or at certain linking groups ofan oligomeric compound.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligomeric compound or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligomeric compound or region thereof. The nucleosides of such anoligomeric compound may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleoside have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substituent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentinvention have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms differs from the atomor a group of atoms normally present in the named functional group. Incertain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino(═NR_(bb)), amido (—C(O)N(R_(bb))(R_(cc)) or—N(R_(bb))C(O)R), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))—(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S—(O)₂R_(bb)).Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, anoptionally linked chemical functional group or a further substituentgroup with a preferred list including without limitation, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl. Selected substituentswithin the compounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or poly-cyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

Oligomeric Compounds

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, such oligomeric compounds compriseoligonucleotides optionally comprising one or more conjugate and/orterminal groups. In certain embodiments, an oligomeric compound consistsof an oligonucleotide. In certain embodiments, oligonucleotides compriseone or more chemical modifications. Such chemical modifications includemodifications one or more nucleoside (including modifications to thesugar moiety and/or the nucleobase) and/or modifications to one or moreinternucleoside linkage.

Certain Sugar Moieties

In certain embodiments, oligomeric compounds of the invention compriseone or more modified nucleosides comprising a modified sugar moiety.Such oligomeric compounds comprising one or more sugar-modifiednucleosides may have desirable properties, such as enhanced nucleasestability or increased binding affinity with a target nucleic acidrelative to oligomeric compounds comprising only nucleosides comprisingnaturally occurring sugar moieties. In certain embodiments, modifiedsugar moieties are substituted sugar moieties. In certain embodiments,modified sugar moieties are bicyclic or tricyclic sugar moieties. Incertain embodiments, modified sugar moieties are sugar surrogates. Suchsugar surrogates may comprise one or more substitutions corresponding tothose of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more substituent, including but not limitedto substituents at the 2′ and/or 5′ positions. Examples of sugarsubstituents suitable for the 2′-position, include, but are not limitedto: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE”). Incertain embodiments, sugar substituents at the 2′ position is selectedfrom allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, O—C₁-C₁₀substituted alkyl; O—C₁-C₁₀ alkoxy; O—C₁-C₁₀ substituted alkoxy, OCF₃,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where eachRm and Rn is, independently, H or substituted or unsubstituted C₁-C₁₀alkyl. Examples of sugar substituents at the 5′-position, include, butare not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. Incertain embodiments, substituted sugars comprise more than onenon-bridging sugar substituent, for example, 2′-F-5′-methyl sugarmoieties (see, e.g., PCT International Application WO 2008/101157, foradditional 5′, 2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, O—C₁-C₁₀ alkoxy; O—C₁-C₁₀ substituted alkoxy, SH, CN, OCN,CF₃, OCF₃, O-alkyl, S-alkyl, N(R_(m))-alkyl; O-alkenyl, S-alkenyl, orN(R_(m))-alkenyl; O-alkynyl, S-alkynyl, N(R_(m))-alkynyl;O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl,O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)),where each R_(m) and R_(n) is, independently, H, an amino protectinggroup or substituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituentgroups can be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′- CH₂-2′,4′—(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, andanalogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15,2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g.,WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, e.g., WO2008/150729, published Dec. 11, 2008);4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004);4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett.,1998, 8, 2219-2222; Singh et al., J Org. Chem., 1998, 63, 10035-10039;Srivastava et al., J Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007);Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braaschet al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol.Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748,6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S.Patent Publication Nos. US2004/0171570, US2007/0287831, andUS2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574,61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and61/099,844; and PCT International Applications Nos. PCT/US2008/064591,PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surrogates comprise a 4′-sulfur atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran. Such tetrahydropyrans may befurther modified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group; q₁, q₂, q₃, q₄, q₅, q₆ and q₇ areeach, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systemsare known in the art that can be used to modify nucleosides (see, e.g.,review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002,10, 841-854).

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following structure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

Certain Nucleobases

In certain embodiments, nucleosides of the present invention compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present invention comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

Certain Internucleoside Linkages

In certain embodiments, the present invention provides oligomericcompounds comprising linked nucleosides. In such embodiments,nucleosides may be linked together using any internucleoside linkage.The two main classes of internucleoside linking groups are defined bythe presence or absence of a phosphorus atom. Representative phosphoruscontaining internucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of the oligomericcompound. In certain embodiments, internucleoside linkages having achiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), a or 3 such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Certain Motifs

In certain embodiments, the present invention provides oligomericcompounds comprising oligonucleotides. In certain embodiments, sucholigonucleotides comprise one or more chemical modification. In certainembodiments, chemically modified oligonucleotides comprise one or moremodified nucleosides. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleosides comprisingmodified sugars. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleosides comprisingone or more modified nucleobases. In certain embodiments, chemicallymodified oligonucleotides comprise one or more modified internucleosidelinkages. In certain embodiments, the chemically modifications (sugarmodifications, nucleobase modifications, and/or linkage modifications)define a pattern or motif. In certain embodiments, the patterns ofchemical modifications of sugar moieties, internucleoside linkages, andnucleobases are each independent of one another. Thus, anoligonucleotide may be described by its sugar modification motif,internucleoside linkage motif and/or nucleobase modification motif (asused herein, nucleobase modification motif describes the chemicalmodifications to the nucleobases independent of the sequence ofnucleobases).

Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar modification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar modification motif, which comprises twoexternal regions or “wings” and an internal region or “gap.” The threeregions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar modification motifs of the two wings are the sameas one another (symmetric gapmer). In certain embodiments, the sugarmodification motifs of the 5′-wing differs from the sugar modificationmotif of the 3′-wing (asymmetric gapmer). In certain embodiments,oligonucleotides comprise 2′-MOE modified nucleosides in the wings and2′-F modified nucleosides in the gap.

In certain embodiments, oligonucleotides are fully modified. In certainsuch embodiments, oligonucleotides are uniformly modified. In certainembodiments, oligonucleotides are uniform 2′-MOE. In certainembodiments, oligonucleotides are uniform 2′-F. In certain embodiments,oligonucleotides are uniform morpholino. In certain embodiments,oligonucleotides are uniform BNA. In certain embodiments,oligonucleotides are uniform LNA. In certain embodiments,oligonucleotides are uniform cEt.

In certain embodiments, oligonucleotides comprise a uniformly modifiedregion and additional nucleosides that are unmodified or differentlymodified. In certain embodiments, the uniformly modified region is atleast 5, 10, 15, or 20 nucleosides in length. In certain embodiments,the uniform region is a 2′-MOE region. In certain embodiments, theuniform region is a 2′-F region. In certain embodiments, the uniformregion is a morpholino region. In certain embodiments, the uniformregion is a BNA region. In certain embodiments, the uniform region is aLNA region. In certain embodiments, the uniform region is a cEt region.

In certain embodiments, the oligonucleotide does not comprise more than4 contiguous unmodified 2′-deoxynucleosides. In certain circumstances,antisense oligonucleotides comprising more than 4 contiguous2′-deoxynucleosides activate RNase H, resulting in cleavage of thetarget RNA. In certain embodiments, such cleavage is avoided by nothaving more than 4 contiguous 2′-deoxynucleosides, for example, wherealteration of splicing and not cleavage of a target RNA is desired.

Certain Splicing Motifs

In certain embodiments, oligonucleotides have a certain modificationpattern and/or motif designed to alter the splicing of certain nucleicacid transcripts. In certain embodiments, oligonucleotides have acertain modification pattern and/or motif designed to alter the splicingof certain pre-mRNA transcripts. In certain embodiments,oligonucleotides have a certain modification pattern and/or motifdesigned in such a fashion that the oligonucleotide will not recruitRNase H once bound to a target nucleic acid transcript. For example, incertain such embodiments, an oligonucleotide may have one or more sugarmodifications placed throughout the oligonucleotide so as to have nosegment comprising more than 4 contiguous 2′-deoxynucleosides. Incertain such embodiments, an oligonucleotide may have one or more sugarmodifications placed throughout the oligonucleotide so as to have nosegment comprising more than 3 contiguous 2′-deoxynucleosides. Incertain such embodiments, an oligonucleotide may have one or more sugarmodifications placed throughout the oligonucleotide so as to have nosegment comprising more than 2 contiguous 2′-deoxynucleosides.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a Ad₂Ad₂Ad₂Ad₂Ad₂A motif, wherein each Aindependently comprises a bicyclic modification selected from among LNAand cEt and each d comprises a 2′-deoxynucleoside.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a AAddAddAddAddAddAA motif, wherein each Aindependently comprises a bicyclic modification selected from among LNAand cEt and each d comprises a 2′-deoxynucleoside.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a AABBABBABBABBABBAB motif, wherein each Aindependently comprises a bicyclic modification selected from among LNAand cEt and each B independently comprises a 2′-modification selectedfrom among a 2′-OMe, 2′-F, or 2′-MOE modification. In certainembodiments, the oligonucleotide compound comprises a modifiedoligonucleotide consisting of 8 to 30 linked nucleosides and having anucleobase sequence comprising a complementary region comprising atleast 8 contiguous nucleobases complementary to a target region of equallength of a target nucleic acid, wherein the modified oligonucleotidecomprises a AddAddAddAddAddA motif wherein each A independentlycomprises a bicyclic modification selected from among LNA and cEt andeach d comprises a 2′-deoxynucleoside.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a keekeekeekeekeek motif, wherein each kcomprises a cEt modification and each e comprises a 2′-MOE modification.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a kd₂kd₂kd₂kd₂kd₂k motif, wherein each kcomprises a cEt modification and each d comprises a 2′-deoxynucleoside.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a kkddkddkddkddkddkk motif, wherein each kcomprises a cEt modification and each d comprises a 2′-deoxynucleoside.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a kkeekeekeekeekeeke motif, wherein each kcomprises a cEt modification and each e comprises a 2′-MOE modification.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a kddkddkddkddkddk motif wherein each kcomprises a cEt modification and each d comprises a 2′-deoxynucleoside.

In certain embodiments, the oligonucleotide compound comprises amodified oligonucleotide consisting of 8 to 30 linked nucleosides andhaving a nucleobase sequence comprising a complementary regioncomprising at least 8 contiguous nucleobases complementary to a targetregion of equal length of a target nucleic acid, wherein the modifiedoligonucleotide comprises a keekeekeekeekeek motif, wherein each kcomprises a cEt modification and each e comprises a 2′-MOE modification.

Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif, as described above for sugar modification motif. In suchembodiments, the internucleoside linkages in each of two wing regionsare different from the internucleoside linkages in the gap region. Incertain embodiments the internucleoside linkages in the wings arephosphodiester and the internucleoside linkages in the gap arephosphorothioate. The sugar modification motif is independentlyselected, so such oligonucleotides having a gapped internucleosidelinkage motif may or may not have a gapped sugar modification motif andif it does have a gapped sugar motif, the wing and gap lengths may ormay not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present invention comprise a region of uniformlymodified internucleoside linkages. In certain such embodiments, theoligonucleotide comprises a region that is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide is uniformly linked by phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate and at least oneinternucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certain suchembodiments, nucleobase modifications are arranged in a gapped motif. Incertain embodiments, nucleobase modifications are arranged in analternating motif. In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

Certain Overall Lengths

In certain embodiments, the present invention provides oligomericcompounds including oligonucleotides of any of a variety of ranges oflengths. In certain embodiments, the invention provides oligomericcompounds or oligonucleotides consisting of X to Y linked nucleosides,where X represents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≤Y. For example, in certainembodiments, the invention provides oligomeric compounds which compriseoligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linkednucleosides. In embodiments where the number of nucleosides of anoligomeric compound or oligonucleotide is limited, whether to a range orto a specific number, the oligomeric compound or oligonucleotide may,nonetheless further comprise additional other substituents. For example,an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotideshaving 31 nucleosides, but, unless otherwise indicated, such anoligonucleotide may further comprise, for example one or moreconjugates, terminal groups, or other substituents. In certainembodiments, a gapmer oligonucleotide has any of the above lengths. Incertain embodiments, an antisense oligonucleotide has any of the abovelengths.

One of skill in the art will appreciate that certain lengths may not bepossible for certain motifs. For example: a gapmer having a 5′-wingregion consisting of four nucleotides, a gap consisting of at least sixnucleotides, and a 3′-wing region consisting of three nucleotides cannothave an overall length less than 13 nucleotides. Thus, one wouldunderstand that the lower length limit is 13 and that the limit of 10 in“10-20” has no effect in that embodiment.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range. For example, an oligonucleotide consisting of20-25 linked nucleosides comprising a 5′-wing consisting of 5 linkednucleosides; a 3′-wing consisting of 5 linked nucleosides and a centralgap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap(before reaching the overall length limitation of 25). Such additionalnucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing.

Certain Oligonucleotides

In certain embodiments, oligonucleotides of the present invention arecharacterized by their sugar motif, internucleoside linkage motif,nucleobase modification motif and overall length. In certainembodiments, such parameters are each independent of one another. Thus,each internucleoside linkage of an oligonucleotide having a gapmer sugarmotif may be modified or unmodified and may or may not follow the gapmermodification pattern of the sugar modifications. Thus, theinternucleoside linkages within the wing regions of a sugar-gapmer maybe the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region. Likewise,such sugar-gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.Herein if a description of an oligonucleotide or oligomeric compound issilent with respect to one or more parameter, such parameter is notlimited. Thus, an oligomeric compound described only as having a gapmersugar motif without further description may have any length,internucleoside linkage motif, and nucleobase modification motif. Unlessotherwise indicated, all chemical modifications are independent ofnucleobase sequence.

Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamics, pharmacokinetics, stability, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligomericcompound, such as an oligonucleotide. Conjugate groups includes withoutlimitation, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groupshave been described previously, for example: cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

In certain embodiments, conjugate groups are directly attached tooligonucleotides in oligomeric compounds. In certain embodiments,conjugate groups are attached to oligonucleotides by a conjugate linkinggroup. In certain such embodiments, conjugate linking groups, including,but not limited to, bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Conjugate linkinggroups are useful for attachment of conjugate groups, such as chemicalstabilizing groups, functional groups, reporter groups and other groupsto selective sites in a parent compound such as for example anoligomeric compound. In general a bifunctional linking moiety comprisesa hydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such aschemical functional group or a conjugate group. In some embodiments, theconjugate linker comprises a chain structure or an oligomer of repeatingunits such as ethylene glycol or amino acid units. Examples offunctional groups that are routinely used in a bifunctional linkingmoiety include, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties includepyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group.

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, oligomeric compounds comprise anoligonucleotide. In certain embodiments, an oligomeric compoundcomprises an oligonucleotide and one or more conjugate and/or terminalgroups. Such conjugate and/or terminal groups may be added tooligonucleotides having any of the chemical motifs discussed above.Thus, for example, an oligomeric compound comprising an oligonucleotidehaving region of alternating nucleosides may comprise a terminal group.

Antisense Compounds

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. Such antisense compounds are capable ofhybridizing to a target nucleic acid, resulting in at least oneantisense activity. In certain embodiments, antisense compoundsspecifically hybridize to one or more target nucleic acid. In certainembodiments, a specifically hybridizing antisense compound has anucleobase sequence comprising a region having sufficientcomplementarity to a target nucleic acid to allow hybridization andresult in antisense activity and insufficient complementarity to anynon-target so as to avoid non-specific hybridization to any non-targetnucleic acid sequences under conditions in which specific hybridizationis desired (e.g., under physiological conditions for in vivo ortherapeutic uses, and under conditions in which assays are performed inthe case of in vitro assays).

In certain embodiments, the present invention provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

In certain embodiments antisense compounds and antisenseoligonucleotides comprise single-strand compounds. In certainembodiments antisense compounds and antisense oligonucleotides comprisedouble-strand compounds.

Certain Pathways and Mechanisms Associated with Fibrosis

TGFβ1 and its associated pathways contribute to many processesassociated with wound healing and tissue repair. After an injury, TGFβ1contributes to the healing and restoration of normal tissue by, amongother things, stimulating the production of certain extracellular matrixproteins and inhibiting the degradation of certain matrix proteins. Incertain embodiments, TGFβ1 stimulates the production of fibronectin. Incertain embodiments, TGFβ1 stimulates the production of both the EDA+and EDA− isoforms of fibronectin. In certain embodiments, excessiveamounts of the EDA+ fibronectin isoform causes tissue fibrosis. Incertain embodiments, excessive tissue fibrosis induced by TGFβ1/EDA+impairs normal organ function, impairs cellular function, and/or causescells to change or lose their phenotype. In certain embodiments, therelease and/or activation of TGFβ1 causes the formation of fibrosis andconsequent changes in cell phenotype. In certain embodiments, changes incell phenotype due to fibrosis include, but are not limited to,modulation of cadherin expression, induction of α Smooth Muscle Actin(αSMA), alteration of cortical f-actin localization, induction ofconnexin 43 (Cx 43) expression, alteration of vimentin, alteration oftight junction protein ZO-1, and/or increased secretion of MMP2 & MMP9.In certain embodiments, the release and/or activation of TGFβ1 causes aloss of cell phenotype. In certain embodiments, the loss of cellphenotype due to fibrosis impairs the structure or function of a cell.In certain embodiments, the loss of cell phenotype due to fibrosisdestroys the function of a cell.

In certain embodiments, it is therefore desirable to reduce fibrosiswithout affecting the healing and/or restoration process. In certainembodiments, it is therefore desirable to reduce the formation offibrosis in a cell without reducing or altering the amount and/oractivity of TGFβ1 in the cell. In certain embodiments, it is thereforedesirable to reduce the amount of EDA+ fibronectin in a cell withoutreducing or altering the amount of EDA− fibronectin in the cell. Incertain embodiments, the reduction of the amount of EDA+ fibronectin ina cell in response to TGFβ1 will result in wound healing and tissuerepair without incurring excessive fibrosis. In certain embodiments, theselective reduction of the amount of EDA+ fibronectin in a cell,relative to the amount of EDA− fibronectin in the cell in the responseto TGFβ1 will stimulate wound healing and tissue repair withoutincurring changes in cell phenotype associated with fibrosis. In certainembodiments, the reduction of the amount of EDA+ fibronectin in a cell,relative to the amount of EDA− fibronectin in the cell in response toTGFβ1 will stimulate wound healing and tissue repair without incurringthe loss of cell phenotype due to fibrosis.

In certain embodiments, it is desirable to reverse the formation offibrosis in a cell without reducing or altering the wound healingfunction of TGFβ1 in the cell. In certain embodiments, it is thereforedesirable to reverse the changes caused by fibrosis in the phenotype ofa cell without reducing or altering the wound healing function of TGFβ1in the cell. In certain embodiments, it is therefore desirable toreverse the loss of phenotype in a cell caused by fibrosis withoutreducing or altering the wound healing function of TGFβ1 in the cell.

Certain Target Nucleic Acids and Mechanisms

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid. In certain embodiments, the target nucleic acid is anendogenous RNA molecule. In certain embodiments, the target nucleic acidis a pre-mRNA. In certain embodiments, the target nucleic acid is afibronectin transcript. In certain embodiments, the target RNA is afibronectin pre-mRNA.

In certain embodiments, an antisense compound is complementary to aregion of fibronectin pre-mRNA. In certain embodiments, an antisensecompound is complementary within a region of fibronectin pre-mRNAcomprising an exon encoding EDA. In certain embodiments, an antisensecompound is complementary to a region of fibronectin pre-mRNA comprisingan intron-exon splice junction. In certain embodiments, an antisensecompound is complementary to a region of fibronectin pre-mRNA comprisingthe intron-exon splice junction adjacent to the EDA exon. In certainembodiments, an antisense compound is complementary within a region offibronectin pre-mRNA consisting of an exon encoding EDA. In certainembodiments, an antisense compound is complementary within a region offibronectin pre-mRNA comprising an exonic splicing silencer within anexon encoding EDA. In certain embodiments, an antisense compound iscomplementary within a region of fibronectin pre-mRNA comprising anexonic splicing enhancer within an exon encoding EDA.

In certain embodiments, an antisense compound comprises a modifiedoligonucleotide consisting of 8 to 30 linked nucleosides and having anucleobase sequence comprising a complementary region comprising atleast 8 contiguous nucleobases complementary to a target region of equallength of a fibronectin transcript. In certain embodiments, the targetregion is within nucleobase 55469 and nucleobase 55790 of SEQ ID NO.: 1.In certain embodiments, the target region is within nucleobase 55469 andnucleobase 55511 of SEQ ID NO.: 1. In certain embodiments, the targetregion is within nucleobase 55511 and nucleobase 55732 of SEQ ID NO.: 1.In certain embodiments, the target region is within nucleobase 55732 andnucleobase 55790 of SEQ ID NO.: 1.

In certain embodiments, an antisense oligonucleotide modulates splicingof a pre-mRNA. In certain embodiments, an antisense oligonucleotidemodulates splicing a fibronectin pre-mRNA. In certain embodiments, anantisense oligonucleotide increases the amount of fibronectin mRNA. Incertain embodiments, an antisense oligonucleotide increases the amountof EDA− fibronectin mRNA. In certain embodiments, an antisenseoligonucleotide decreases the amount of EDA+ fibronectin mRNA. Incertain embodiments, an antisense oligonucleotide decreases the amountof EDA+ fibronectin mRNA in the presence of TGFβ1. In certainembodiments, an antisense oligonucleotide decreases the amount of EDA+fibronectin mRNA in a cell without substantially affecting the healingand/or restoration functions of the cell.

In certain embodiments, an antisense oligonucleotide alters the ratio ofEDA+/EDA− fibronectin. In certain embodiments, an antisenseoligonucleotide increases the ratio of EDA+/EDA− fibronectin. In certainembodiments, it is desirable to increase the ratio of EDA+/EDA−fibronectin to create a fibrosis model. In certain embodiments, it isdesirable to increase the ratio of EDA+/EDA− fibronectin to create afibrosis phenotype. In certain embodiments, it is desirable to increasethe ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create afibrosis model. In certain embodiments, it is desirable to increase theratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create afibrosis phenotype. In certain embodiments, it is desirable to increasethe ratio of EDA+/EDA− fibronectin to create a fibrosis mouse model. Incertain embodiments, it is desirable to increase the ratio of EDA+/EDA−fibronectin to create a fibrosis mouse phenotype. In certainembodiments, it is desirable to increase the ratio of EDA+/EDA−fibronectin in the presence of TGFβ1 to create a fibrosis mouse model.In certain embodiments, it is desirable to increase the ratio ofEDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosismouse phenotype.

In certain embodiments, an antisense oligonucleotide alters the ratio ofEDA+/EDA− fibronectin. In certain embodiments, an antisenseoligonucleotide decreases the ratio of EDA+/EDA− fibronectin. In certainembodiments, it is desirable to decrease the ratio of EDA+/EDA−fibronectin to create a fibrosis model. In certain embodiments, it isdesirable to decrease the ratio of EDA+/EDA− fibronectin to create afibrosis phenotype. In certain embodiments, it is desirable to decreasethe ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create afibrosis model. In certain embodiments, it is desirable to decrease theratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create afibrosis phenotype. In certain embodiments, it is desirable to decreasethe ratio of EDA+/EDA− fibronectin to create a fibrosis mouse model. Incertain embodiments, it is desirable to decrease the ratio of EDA+/EDA−fibronectin to create a fibrosis mouse phenotype. In certainembodiments, it is desirable to decrease the ratio of EDA+/EDA−fibronectin in the presence of TGFβ1 to create a fibrosis mouse model.In certain embodiments, it is desirable to decrease the ratio ofEDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosismouse phenotype.

In certain embodiments, an antisense oligonucleotide alters the ratio ofEDA−/EDA+ fibronectin. In certain embodiments, an antisenseoligonucleotide increases the ratio of EDA−/EDA+ fibronectin. In certainembodiments, it is desirable to increase the ratio of EDA−/EDA+fibronectin to create a fibrosis model. In certain embodiments, it isdesirable to increase the ratio of EDA−/EDA+ fibronectin to create afibrosis phenotype. In certain embodiments, it is desirable to increasethe ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create afibrosis model. In certain embodiments, it is desirable to increase theratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create afibrosis phenotype. In certain embodiments, it is desirable to increasethe ratio of EDA−/EDA+ fibronectin to create a fibrosis mouse model. Incertain embodiments, it is desirable to increase the ratio of EDA−/EDA+fibronectin to create a fibrosis mouse phenotype. In certainembodiments, it is desirable to increase the ratio of EDA−/EDA+fibronectin in the presence of TGFβ1 to create a fibrosis mouse model.In certain embodiments, it is desirable to increase the ratio ofEDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosismouse phenotype.

In certain embodiments, an antisense oligonucleotide alters the ratio ofEDA−/EDA+ fibronectin. In certain embodiments, an antisenseoligonucleotide decreases the ratio of EDA−/EDA+ fibronectin. In certainembodiments, it is desirable to decrease the ratio of EDA−/EDA+fibronectin to create a fibrosis model. In certain embodiments, it isdesirable to decrease the ratio of EDA−/EDA+ fibronectin to create afibrosis phenotype. In certain embodiments, it is desirable to decreasethe ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create afibrosis model. In certain embodiments, it is desirable to decrease theratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create afibrosis phenotype. In certain embodiments, it is desirable to decreasethe ratio of EDA−/EDA+ fibronectin to create a fibrosis mouse model. Incertain embodiments, it is desirable to decrease the ratio of EDA−/EDA+fibronectin to create a fibrosis mouse phenotype. In certainembodiments, it is desirable to decrease the ratio of EDA−/EDA+fibronectin in the presence of TGFβ1 to create a fibrosis mouse model.In certain embodiments, it is desirable to decrease the ratio ofEDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosismouse phenotype.

Certain Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active antisense oligomericcompound.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present invention provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human.

In certain embodiments, the present invention provides methods ofadministering a pharmaceutical composition comprising an oligomericcompound of the present invention to an animal. Suitable administrationroutes include, but are not limited to, oral, rectal, transmucosal,intestinal, enteral, topical, suppository, through inhalation,intrathecal, intracerebroventricular, intraperitoneal, intranasal,intraocular, intratumoral, and parenteral (e.g., intravenous,intramuscular, intramedullary, and subcutaneous). In certainembodiments, pharmaceutical intrathecals are administered to achievelocal rather than systemic exposures. For example, pharmaceuticalcompositions may be injected directly in the area of desired effect(e.g., into the eyes, ears).

In certain embodiments, a pharmaceutical composition is administered toan animal having at least one symptom associated with fibrosis. Incertain embodiments, such administration results in amelioration of atleast one symptom. In certain embodiments, administration of apharmaceutical composition to an animal results in a decrease of EDA+fibronectin mRNA in a cell of the animal. In certain embodiments, suchadministration results in an increase in EDA− fibronectin mRNA. Incertain embodiments, such administration results in a decrease in EDA+fibronectin protein and an increase EDA− fibronectin protein. In certainembodiments, a fibronectin protein lacking EDA amino acids is preferredover a fibronectin protein having EDA amino acids. In certainembodiments, the administration of certain antisense oligonucleotidesdelays the onset of fibrosis. In certain embodiments, the administrationof certain antisense oligonucleotides slows the progression of fibrosis.In certain embodiments, the administration of certain antisenseoligonucleotides prevents the formation of fibrosis. In certainembodiments, the administration of certain antisense oligonucleotidesreverses fibrosis. In certain embodiments, the administration of certainantisense oligonucleotides rescues cellular phenotype. In certainembodiments, the administration of certain antisense oligonucleotidesrescues cellular morphology.

NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligomeric compound having the nucleobase sequence “ATCGATCG”encompasses any oligomeric compounds having such nucleobase sequence,whether modified or unmodified, including, but not limited to, suchcompounds comprising RNA bases, such as those having sequence “AUCGAUCG”and those having some DNA bases and some RNA bases such as “AUCGATCG”and oligomeric compounds having other modified or naturally occurringbases, such as “AT^(me)CGAUCG,” wherein ^(me)C indicates a cytosine basecomprising a methyl group at the 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the presentinvention and are not limiting. Moreover, where specific embodiments areprovided, the inventors have contemplated generic application of thosespecific embodiments. For example, disclosure of an oligonucleotidehaving a particular motif provides reasonable support for additionaloligonucleotides having the same or similar motif. And, for example,where a particular high-affinity modification appears at a particularposition, other high-affinity modifications at the same position areconsidered suitable, unless otherwise indicated.

Example 1: In Vitro Screening of Human Fibronectin Splicing withAntisense Oligonucleotides in HKC-8 Cells

Antisense oligonucleotides were designed targeting a fibronectin nucleicacid and were tested for their effects on the alternative splicing ofthe fibronectin gene sequence in vitro. The newly designed antisenseoligonucleotides in Table 1 were designed as uniform MOEoligonucleotides. Each nucleoside in the oligonucleotide has a 2′-MOEmodification. The internucleoside linkages throughout eacholigonucleotide are phosphorothioate (P═S) linkages. All cytosineresidues throughout each oligonucleotide are 5-methylcytosines. “Startsite” indicates the 5′-most nucleoside to which the oligonucleotide istargeted in the human gene sequence. “Stop site” indicates the 3′-mostnucleoside to which the oligonucleotide is targeted human gene sequence.Each oligonucleotide listed in Table 1 is targeted to SEQ ID NO: 1 (thecomplement of GENBANK Accession No. NT_005403.14 truncated fromnucleotides 66434501 to 66510708). ISIS 141923 (CCTTCCCTGAAGGTTCCTCC(SEQ ID NO: 25), no known human target) was used as a negative control.

Cultured HKC-8 cells, which are SV40-transformed human proximal tubularcells, were transfected using 3 μL LipofectAMINE2000®/mL OptiMEM with200 nM antisense oligonucleotide. After a treatment period ofapproximately 4 hours, the medium was removed and new medium was added,left in culture overnight. RNA was isolated from the cells and the ratioof Extra Domain A positive fibronectin (EDA⁺FN) to EDA negativefibronectin (EDA-FN) was measured by conventional PCR. Human primerswith forward sequence GGAGAGAGTCAGCCTCTGGTTCAG, designated herein as SEQID NO: 2; reverse sequence TGTCAACTGGGCGCTCAGGCTTGTG, designated hereinas SEQ ID NO: 3) was used to measure mRNA levels. To compare theefficacy of antisense treatments performed in different experiments andallow for inter-assay variability, ratios were indexed on thecorresponding negative control. Results are presented in Table 1 anddemonstrate that treatment with antisense oligonucleotides targeted tothe EDA region of fibronectin resulted in decreased expression of theEDA⁺FN isoform compared to the negative control. ‘null’ indicates thatthe EDA⁺ band was undetectable for that sample.

TABLE 1 Ratio of EDA ⁺FN to EDA ⁻FN in HKC-8 cells after treatment withantisense oligonucleotides targeted to SEQ ID NO: 1 SEQ ID NO: 1SEQ ID NO: 1 ISIS Start Stop EDA ⁺FN/ SEQ ID NO Site Site SequenceEDA ⁻FN NO 511399 55469 55483 GCAAATTAATGGTAA 0.07  5 511400 55473 55487TTAGGCAAATTAATG 0.07  6 511401 55477 55491 TCTGTTAGGCAAATT 0.05  7511402 55480 55494 ATGTCTGTTAGGCAA 0.09  8 511403 55483 55497TCAATGTCTGTTAGG 0.04  9 511404 55486 55500 CGATCAATGTCTGTT 0.04 10511405 55489 55503 GGGCGATCAATGTCT 0.03 11 511406 55493 55507TTTAGGGCGATCAAT 0.03 12 511407 55497 55511 GTCCTTTAGGGCGAT 0.4 13 51140855732 55746 CCAATCAGGGGCTGG 0.02 14 511409 55736 55750 GGTTCCAATCAGGGGnull 15 511410 55740 55754 ACTGGGTTCCAATCA 0.01 16 511411 55743 55757TGGACTGGGTTCCAA 0.02 17 511412 55746 55760 CTGTGGACTGGGTTC null 18511413 55749 55763 TACCTGTGGACTGGG 0.06 19 511414 55752 55766ATATACCTGTGGACT 0.04 20 511415 55756 55770 AACCATATACCTGTG 0.05 21511416 55760 55774 AATTAACCATATACC 0.15 22 511417 55482 55499GATCAATGTCTGTTAGGC 0.12 23 511418 55744 55761 CCTGTGGACTGGGTTCCA null 24141923 n/a n/a CCTTCCCTGAAGGTTCCTCC 1.00 25

Example 2: In Vitro Screening of Human Fibronectin Splicing withAntisense Oligonucleotides in Primary Human Proximal Tubular Cells

The antisense oligonucleotides described in Example 1 were also testedfor their effects on the alternative splicing of the fibronectin genesequence in primary human proximal tubular cells (PTEC). Cultured PTECcells were transfected using 2 μL LipofectAMINE2000®/mL OptiMEM with 100nM antisense oligonucleotide for 4 hours, the medium was removed and newmedium added, left in culture overnight, and then treated with 0.1% BSA(vehicle) or 2.5 ng/mL TGFβ1 in 0.1% BSA for 24 hrs. RNA was isolatedfrom the cells and levels were measured by conventional PCR. The ratioof EDA⁺FN to EDA⁻ FN for the given oligonucleotide-treated cells to theratio for the negative control-treated cells was calculated. Results arepresented in Table 2 and indicate that treatment with antisenseoligonucleotides targeted to the EDA region of fibronectin resulted indecreased expression of the EDA⁺FN isoform compared to the negativecontrol, even after induction with TGFβ1. ‘null’ indicates that the EDA⁺band was undetectable for that sample.

TABLE 2 Ratio of EDA⁺FN to EDA⁻FN in PTEC cells after treatment withantisense oligonucleotides EDA⁺FN/ EDA⁻FN EDA⁺FN/ ISIS (without EDA⁻FN(with SEQ ID NO TGFβ1) TGFβ1) NO 511399 0.13 0.17 5 511400 0.23 0.26 6511401 0.14 0.26 7 511402 0.20 0.27 8 511403 0.13 0.23 9 511404 0.090.11 10 511405 0.06 0.08 11 511406 0.03 0.04 12 511407 0.41 0.66 13511408 null null 14 511409 null null 15 511410 null null 16 511411 0.100.09 17 511412 null null 18 511413 0.08 0.09 19 511414 0.15 0.13 20511415 0.34 0.42 21 511416 0.62 0.97 22 511417 0.17 0.20 23 511418 nullnull 24 141923 1.00 1.61 25

Example 3: Effect of Antisense Oligonucleotides Targeting the EDA Regionof Fibronectin on TGFβ1 Induction of EDA⁺FN mRNA Expression in PrimaryHuman Proximal Tubular Cells

Antisense oligonucleotides selected from the studies described abovewere tested for their effects on the alternative splicing of thefibronectin gene sequence in primary human proximal tubular cells (PTEC)treated with TGFβ1. One set of cultured PTEC cells were transfectedusing 2 μL LipofectAMINE2000®/mL OptiMEM with 100 nM antisenseoligonucleotide. These cells were treated for 4 hours with antisenseoligonucleotide; the medium was removed and new medium added; left inculture overnight; and then treated with 0.1% BSA (vehicle) or 2.5 ng/mLTGFβ1 in 0.1% BSA for 24 hrs. These cells were designated as pre-TGFβ1.Another set of cells were first treated with 0.1% BSA (vehicle) or 2.5ng/mL TGFβ1 in 01% BSA for 24 hrs; then transfected using 2 μLLipofectAMINE2000®/mL OptiMEM with 100 nM antisense oligonucleotide for4 h; the medium was removed and new medium added; and then left inculture overnight. These cells were designated as post-TGFβ1. RNA wasisolated from the cells and levels were measured by conventional PCR.The ratio of EDA⁺FN to EDA⁻ FN for the given oligonucleotide-treatedcells to the ratio for the negative control-treated cells wascalculated. Results are presented in Tables 3 and 4, and indicate thattreatment with antisense oligonucleotides targeted to the EDA region offibronectin resulted in decreased expression of the EDA⁺FN isoformcompared to the negative control, irrespective of whether the treatmentwith antisense oligonucleotides took place before or after inductionwith TGFβ1 ‘null’ indicates that the EDA⁺ band was undetectable for thatsample.

TABLE 3 Ratio of EDA⁺FN to EDA⁻FN in pre- TGFβ1 PTEC cells aftertreatment with antisense oligonucleotides EDA⁺FN/ EDA⁺FN/ EDA⁻FN EDA⁻FN(without (with SEQ ID ISIS NO TGFβ1) TGFβ1) NO 511399 0.13 0.17 5 5114030.13 0.23 9 511407 0.41 0.66 13 511408 null null 14 511412 null null 18511416 0.62 0.97 22 141923 1.00 1.61 25

TABLE 4 Ratio of EDA⁺FN to EDA⁻FN in post-TGFβ1 PTEC cells aftertreatment with antisense oligonucleotides EDA⁺FN/ EDA⁺FN/ EDA⁻FN EDA⁻FN(without (with SEQ ID ISIS NO TGFβ1) TGFβ1) NO 511399 0.68 0.75 5 5114030.48 0.30 9 511407 0.68 0.68 13 511408 0.22 0.16 14 511412 null null 18511416 0.79 0.69 22 141923 1.00 1.57 25

Example 4: Effect of Antisense Oligonucleotides Targeting the EDA Regionof Fibronectin on TGFβ1 Induction of EDA⁺FN Protein Expression inPrimary Human Proximal Tubular Cells

The antisense oligonucleotides described above were tested for theireffects on the alternative splicing of the fibronectin gene sequence inprimary human proximal tubular cells (PTEC) treated with TGFβ1. CulturedPTEC cells were transfected using 2 μL LipofectAMINE2000/mL OptiMEM with100 nM antisense oligonucleotide. The cells were treated for 4 hourswith antisense oligonucleotide; the medium was removed and new mediumadded; left in culture overnight; and then treated with 0.1% BSA(vehicle) or 2.5 ng/mL TGFβ1 in 01% BSA for 48 hrs. The cells were lysedin lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X,0.5% sodium deoxycholate, 0.1% SDS; pH 7.2) and protein was extracted asdescribed in Phanish M K et al., Biochem J. 2006 Jan. 15; 393(Pt2):601-7. The protein samples were run on an SDS-PAGE and analyzed viawestern analysis using the anti-Fibronectin antibody [IST-9] (Abcam,ab6328) that reacts with an epitope located in the ED-A sequence ofcellular fibronectin. Results are presented in Table 5, and indicatethat treatment with antisense oligonucleotides targeted to the EDAregion of fibronectin resulted in decreased protein expression of theEDA⁺FN isoform compared to the negative control.

TABLE 5 EDA⁺FN expression in PTEC cells after treatment with antisenseoligonucleotides (densitometric analysis on one Western blot) EDA⁺FNEDA⁺FN (without (with SEQ ID ISIS NO TGFβ1) TGFβ1) NO 511399 0.07 0.03 5511403 0.02 0.04 9 511407 0.13 0.75 13 511408 0.08 0.77 14 511412 0.250.30 18 511416 0.61 0.58 22 141923 0.31 0.74 25

Example 5: Effect of ISIS 511403 on TGFβ1 Induction of Fibrosis inPrimary Human Proximal Tubular Cells

ISIS 511403 was tested for its effect on the alternative splicing of thefibronectin gene sequence in primary human proximal tubular cells (PTEC)treated with TGFβ1. Cultured PTEC cells were treated with 0.1% BSA(vehicle) or with 2.5 ng/mL TGFβ1 in 01% BSA for 24 hrs and thentransfected using 2 μL LipofectAMINE2000®/mL OptiMEM with 100 nM ISIS511403 or 100 nM ISIS 141923 for 24 h. After a recovery period of 24 hin normal growth medium, RNA was isolated and the ratio of EDA⁺FN toEDA^(−□)FN was measured by conventional PCR. In addition, the individualexpressions of EDA⁺FN and EDA^(−□)FN normalized to 18s RNA were alsomeasured (human primer probe set for 18S: forward sequence:GTAACCCGTTGAACCCCATT (SEQ ID NO: 26), reverse sequence:CCATCCAATCGGTAGTAGCG (SEQ ID NO: 27)). In addition, the expression oftotal fibronectin was also measured by quantitative real-time PCR (probeset Hs01549940_ml, Applied Biosystems). The results are presented inTable 6 and indicate that treatment with ISIS 511403 decreased the ratioof EDA⁺FN to EDA⁻FN, decreased expression of EDA⁺FN, increasedexpression of EDA⁻FN, and had no effect on total fibronectin expressioncompared to that of the negative control cells.

TABLE 6 Effect of ISIS 511403 in PTEC cells treated with TGFβ1 withoutwith TGFβ1 TGFβ1 EDA⁺FN/ ISIS 141923 0.90 1.54 EDA^(−□)FN ISIS 5114030.21 0.12 EDA⁺FN/18S ISIS 141923 0.21 0.21 ISIS 511403 0.07 0.68EDA^(−□)FN/18S ISIS 141923 0.23 0.11 ISIS 511403 0.32 0.86 Total ISIS141923 1.00 (0.82-1.23) 3.98 (3.60-4.40) Fibronectin (range) ISIS 5114031.37 (1.17-1.60) 4.14 (4.05-4.23)

The effect of treatment with ISIS 511403 on lactate dehydrogenase (LDH)release by the cells was also measured using the CytoTox 96®Non-Radioactive Cytotoxicity Assay (Promega, G1780). The results arepresented in Table 7 and indicate the decrease in LDH release in cellstreated with ISIS 511403 compared to the cells treated with the negativecontrol. This demonstrates that ISIS 511403 can rescue certainpronounced changes in cell phenotype caused by TGFβ1 induction, e.g. therelease of LDH.

TABLE 7 Effect of ISIS 511403 on LDH release in PTEC cells treated withTGFβ1 Absorbance at 490 nm ISIS 141923 without TGFβ1 0.14 with TGFβ10.12 ISIS 511403 without TGFβ1 0.33 with TGFβ1 0.16

The effect of treatment with ISIS 511403 on αSMA mRNA expression by thecells was also measured by quantitative real-time PCR, using primerprobe set Hs00909449_ml (Applied Biosystems). The results are presentedin Table 8 and indicate the decrease in αSMA in cells treated with ISIS511403 compared to the cells treated with the negative control. Thenumbers in parentheses indicate the range. This demonstrates that ISIS511403 can rescue certain pronounced changes in cell phenotype caused byTGFβ1 induction, e.g. the induction of αSMA.

TABLE 8 Effect of ISIS 511403 on αSMA expression in PTEC cells treatedwith TGFβ1 Fold increase over basal levels ISIS 141923 without TGFβ11.00 (0.79-1.26) with TGFβ1 1.96 (1.70-2.25) ISIS 511403 without TGFβ10.70 (0.59-0.83) with TGFβ1 0.79 (0.68-0.91)

The data presented in Tables 7 and 8 indicate that treatment withantisense oligonucleotides inhibiting the splicing and inclusion of theEDA region of fibronectin resulted in decreased fibrosis in primaryhuman PTEC and therefore have therapeutic benefit in the prevention,treatment, or amelioration of fibrosis.

Additionally, by staining it was observed that prevention of EDAinclusion by treatment with ISIS 511403 resulted in a significantreduction in αSMA compared to treatment with a control (ISIS 141923). Bywestern blot analysis, it was also observed that prevention of EDAinclusion by treatment with ISIS 511403 resulted in reduction insecretion of MMP2 & MMP9. The fold-change in the cell motility marker, S100A4 was measured and is presented in Table 9. Treatment with ISIS511403 resulted in significant reduction in S100A4 in TGF-β-treatedcells.

TABLE 9 Effect of ISIS 511403 on S100A4 expression in PTEC cells treatedwith TGFβ1 Fold increase over basal levels ISIS 141923 without TGFβ11.00 with TGFβ1 1.45 ISIS 511403 without TGFβ1 0.62 with TGFβ1 0.74

By staining and by western blot analysis, it was also observed thatprevention of EDA inclusion by treatment with ISIS 511403 resulted innear complete inhibition of Connexin 43. By staining, it was alsoobserved that prevention of EDA inclusion by treatment with ISIS 511403resulted in a moderate increase in f-actin localization.

Example 6: Design of Antisense Oligonucleotides Targeting Human andMurine Fibronectin

Antisense oligonucleotides were designed targeting a fibronectin nucleicacid. The newly designed chimeric antisense oligonucleotides in Table 10were designed as uniform MOE oligonucleotides. Each nucleoside in theoligonucleotide has a 2′-MOE modification. The internucleoside linkagesthroughout each oligonucleotide are phosphorothioate (P═S) linkages. Allcytosine residues throughout each oligonucleotide are 5-methylcytosines.“Human Start site” indicates the 5′-most nucleoside to which theoligonucleotide is targeted in the human gene sequence. “Murine StartSite” indicates the 5′-most nucleoside to which the oligonucleotide istargeted murine gene sequence. Each oligonucleotide listed in Table 10is targeted to the human fibronectin genomic sequence, SEQ ID NO: 1 (thecomplement of GENBANK Accession No. NT_005403.14 truncated fromnucleotides 66434501 to 66510708) or to murine fibronectin genomicsequence, SEQ ID NO: 29 (the complement of Accession No. NT_039170.2truncated from nucleotides 20696091 to 20764741), or both. Several ofthe oligonucleotides are cross-reactive with human and mouse genesequences. The greater the complementarity between the oligonucleotideand the gene sequence, the more likely the oligonucleotide can targetthe gene sequence. ‘Mismatches’ indicates the number of nucleotides inthe oligonucleotide that are mismatched with the gene sequence. ‘n/a’indicates that the oligonucleotide contains more than 2 mismatches withthe particular gene sequence.

TABLE 10Antisense oligonucleotides targeted to SEQ ID NO: 1 and SEQ ID NO: 29Human Mouse target Target Start Site Mismatches Start Site MismatchesSEQ ID (SEQ ID with SEQ (SEQ ID with SEQ NO of NO: 1) ID NO: 1 SequenceISIS No NO: 29) ID NO: 29) oligo 55458 0 GTAAGAGGTTATGTG 594692 49712 030 55464 0 TTAATGGTAAGAGGT 594693 49718 0 31 55478 0 AATGTCTGTTAGGCAAAT594685 49732 0 32 55479 0 CAATGTCTGTTAGGCAAA 594686 49733 0 33 55480 0TCAATGTCTGTTAGGCAA 594687 49734 0 34 55481 0 AATGTCTGTTAGGCA 59468149735 0 35 55481 0 ATCAATGTCTGTTAGGCA 594688 49735 0 36 55482 0CAATGTCTGTTAGGC 594682 49736 0 37 55483 0 CGATCAATGTCTGTTAGG 59468949737 0 38 55484 0 ATCAATGTCTGTTAG 594683 49738 0 39 55484 0GCGATCAATGTCTGTTAG 594690 49738 0 40 55485 0 GATCAATGTCTGTTA 59468449739 0 41 55485 0 GGCGATCAATGTCTGTTA 594691 49739 0 42 55501 0GCCAGTCCTTTAGGG 594694 49755 0 43 55507 0 GTGAATGCCAGTCCT 594695 49761 044 55513 0 ACATCAGTGAATGCC 594696 49767 0 45 55519 0 ACATCCACATCAGTG594697 49773 0 46 55525 0 GAATCGACATCCACA 594698 49779 0 47 55531 0TTGATGGAATCGACA 594699 49785 0 48 55537 0 GCAATTTTGATGGAA 594700 49791 049 55543 0 TCCCAAGCAATTTTG 594701 49797 0 50 55549 0 GGGCTTTCCCAAGCA594702 49803 0 51 55555 0 CCCTGTGGGCTTTCC 594703 49809 0 52 55561 0ACTTGCCCCTGTGGG 594704 49815 0 53 55567 0 CTGGAAACTTGCCCC 594705 49821 054 55573 0 CTGTACCTGGAAACT 594706 49827 0 55 55579 0 GTCACCCTGTACCTG594707 49833 0 56 55585 0 GAGTAGGTCACCCTG 594708 49839 0 57 55591 0GGGCTCGAGTAGGTC 594709 49845 0 58 55597 0 TCCTCAGGGCTCGAG 594710 49851 059 55603 0 ATTCCATCCTCAGGG 594711 49857 0 60 55609 0 TCATGGATTCCATCC594712 49863 2 61 55615 0 AATAGCTCATGGATT 594713 n/a n/a 62 55621 0GCAGGGAATAGCTCA 594714 49875 2 63 55627 0 TCAGGTGCAGGGAAT 594715 49881 164 55633 0 TCACCATCAGGTGCA 594716 49887 0 65 55639 0 TCTTCTTCACCATCA594717 49893 1 66 55645 0 GCAGTGTCTTCTTCA 594718 49899 1 67 55651 0AGCTCTGCAGTGTCT 594719 49905 1 68 55657 0 CCTTGCAGCTCTGCA 594720 49911 169 55663 0 CTGAGGCCTTGCAGC 594721 49917 1 70 55669 0 CCCGGTCTGAGGCCT594722 49923 2 71 55675 0 TCAGAACCCGGTCTG 594723 49929 2 72 55681 0GTGTACTCAGAACCC 594724 49935 1 73 55687 0 CTGACTGTGTACTCA 594725 49941 074 55693 0 ACCACACTGACTGTG 594726 49947 0 75 55699 0 AAGGCAACCACACTG594727 49953 0 76 55705 0 TCGTGCAAGGCAACC 594728 49959 0 77 55711 0ATATCATCGTGCAAG 594729 49965 0 78 55717 0 CTCTCCATATCATCG 594730 49971 079 55723 0 GGCTGGCTCTCCATA 594731 49977 0 80 55736 1 GATTCCAATCAGGGG594670 49990 0 81 55740 1 ACTGGATTCCAATCA 594671 49994 0 82 55743 1TGGACTGGATTCCAA 594672 49997 0 83 55744 1 CCTGTGGACTGGATTCCA 59467749998 0 84 55746 1 CTGTGGACTGGATTC 594673 50000 0 85 55749 1TACCTGTGGACTGGA 594674 50003 0 86 55756 1 AACGATATACCTGTG 594675 50010 087 55760 2 AATTAATCATAAACC 594676 39337 0 88 55765 0 GGTGCAATTAACCAT594732 n/a n/a 89 55771 0 CCTGGTGGTGCAATT 594733 n/a n/a 90

Example 7: In Vitro Screening of Uniform MOE Antisense OligonucleotidesTargeting Human and/or Mouse Fibronectin in MHT Cells

Some of the antisense oligonucleotides presented in Example 6 weretested for potency in cultured primary PTEC cells. ISIS 511403 was alsoincluded in the study.

Cultured PTEC cells were transfected using 2 μL LipofectAMINE2000®/mLOptiMEM with 100 nM antisense oligonucleotide for 4 hours, the mediumwas removed and new medium added, left in culture overnight, and thentreated with 0.1% BSA (vehicle) or 2.5 ng/mL TGFβ1 in 0.1% BSA for 24hrs. RNA was isolated from the cells and levels of EDA⁺FN mRNA weremeasured by RT-PCR using primer probe sets RTS3963_MGB (forward sequenceGCCTTGCACGATGATATGGA, designated herein as SEQ ID NO: 91; reversesequence TGTGGGTGTGACCTGAGTGAA, designated herein as SEQ ID NO: 92;probe sequence ATTGGAACCCAGTCCAC, designated herein as SEQ ID NO: 93),as well as with primer probe set RTS3964 (forward sequenceGAATCCAAGCGGAGAGAGTCA, designated herein as SEQ ID NO: 94; reversesequence ACATCAGTGAATGCCAGTCCTTT, designated herein as SEQ ID NO: 95;probe sequence TTCAGACTGCAGTAACCAACATTGATCGCC, designated herein as SEQID NO: 96), both of which are designed to the EDA+ variant of the FNmRNA transcript (NM_212478.1, designated herein as SEQ ID NO: 97) andwhich target different regions of the transcript. For data analysis, thelevels of EDA⁺FN mRNA were normalized to the levels of the house-keepinggene, the large ribosomal protein transcript (Human RPLPO, AppliedBiosystems, cat#4333761F). For each antisense oligonucleotide, the ratioof EDA⁺FN to RPLPO in antisense oligonucleotide-treated cells was thennormalized to the ratio of EDA⁺FN mRNA to RPLPO in untreated cells. Theresults are presented in Table 11. The results indicate that treatmentwith antisense oligonucleotides reduced expression of the EDA+transcript compared to untreated cells, both in the presence or absenceof TGFβ1.

TABLE 11 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) in primary PTEC cells without with ISIS No PPset Used TGFβ1 TGFβ1511403 RTS3963_MGB 0.21 0.21 594692 RTS3963_MGB 0.17 0.29 594693RTS3963_MGB 0.21 0.32 594694 RTS3963_MGB 0.02 0.03 594695 RTS3963_MGB0.15 0.24 594696 RTS3963_MGB 0.23 0.41 594697 RTS3963_MGB 0.40 0.70594698 RTS3963_MGB 0.50 0.81 594699 RTS3963_MGB 0.51 0.76 594700RTS3963_MGB 0.13 0.18 594701 RTS3963_MGB 0.33 0.47 594702 RTS3963_MGB0.30 0.49 594703 RTS3963_MGB 0.16 0.32 594704 RTS3963_MGB 0.56 0.73594705 RTS3963_MGB 0.40 0.68 594706 RTS3963_MGB 0.40 0.81 594707RTS3963_MGB 0.29 0.40 594708 RTS3963_MGB 0.06 0.05 594709 RTS3963_MGB0.03 0.02 594710 RTS3963_MGB 0.18 0.16 594711 RTS3963_MGB 0.43 0.27594712 RTS3963_MGB 0.36 0.31 594713 RTS3963_MGB 0.14 0.15 594714RTS3963_MGB 0.06 0.08 594715 RTS3963_MGB 0.05 0.07 594716 RTS3963_MGB0.18 0.16 594717 RTS3963_MGB 0.71 0.68 594718 RTS3963_MGB 0.22 0.33594719 RTS3963_MGB 0.46 0.44 594720 RTS3963_MGB 0.13 0.14 594721RTS3963_MGB 0.14 0.13 594722 RTS3963_MGB 0.07 0.15 594723 RTS3963_MGB0.05 0.05 594724 RTS3963_MGB 0.11 0.17 594725 RTS3963_MGB 0.06 0.07594726 RTS3963_MGB 0.12 0.17 594727 RTS3964 0.06 0.10 594728 RTS39640.03 0.02 594729 RTS3964 0.08 0.06 594730 RTS3964 0.39 0.38 594731RTS3964 0.10 0.07 594732 RTS3963_MGB 0.92 0.79 594733 RTS3963_MGB 0.370.38

Example 8: In Vitro Screening of Uniform MOE Antisense OligonucleotidesTargeting Human and/or Mouse Fibronectin in MHT Cells

Antisense oligonucleotides were designed targeting a fibronectin nucleicacid and were tested for their effects on blocking of splicing in vitro.The newly designed chimeric antisense oligonucleotides in Table 12 weredesigned as uniform MOE oligonucleotides. Each oligonucleotide is 15nucleosides long and each nucleoside in the oligonucleotide has a 2′-MOEmodification. The internucleoside linkages throughout eacholigonucleotide are phosphorothioate (P═S) linkages. All cytosineresidues throughout each oligonucleotide are 5-methylcytosines. “HumanStart site” indicates the 5′-most nucleoside to which theoligonucleotide is targeted in the human gene sequence. “Murine StartSite” indicates the 5′-most nucleoside to which the oligonucleotide istargeted murine gene sequence. Each oligonucleotide listed in Table 12is targeted to the human fibronectin genomic sequence, SEQ ID NO: 1 orto murine fibronectin genomic sequence, SEQ ID NO: 22, or both. Severalof the oligonucleotides are cross-reactive with human and mouse genesequences. The greater the complementarity between the oligonucleotideand the gene sequence, the more likely the oligonucleotide can targetthe gene sequence. ‘Mismatches’ indicates the least number ofnucleotides in the oligonucleotide that are mismatched with the genesequence; the antisense oligonucleotide may target the gene sequencewith more mismatches. ‘n/a’ indicates that the antisense oligonucleotidehas more than 3 mismatches with the particular gene sequence.

Cultured MHT cells, a mouse hepatocellular carcinoma cell line (Koller,E. et al., Nucleic Acids Research, 2011, 1-13), were transfected using 5μl LipofectAMINE2000®/mL OptiMEM with 50 nM antisense oligonucleotide.After a treatment period of approximately 4 hours, the medium wasremoved and new medium was added, and the cells were left in cultureovernight. RNA was isolated from the cells and measured by RT-PCR.EDA⁺FN mRNA expression was measured with mouse primer probe set LTS01050(forward sequence AAACTGCAGTGACCAACATTGATC, designated herein as SEQ IDNO: 98; reverse sequence CTTGCCCCTGTGGGCTTT, designated herein as SEQ IDNO: 99; probe sequence CTGATGTGGATGTCGATT, designated herein as SEQ IDNO: 100), as well as with LTS01052 (forward sequence GCCAGCCCCTGATTGGA,designated herein as SEQ ID NO: 101; reverse sequenceCCGGTAGCCAGTGAGCTGAA, designated herein as SEQ ID NO: 102; probesequence CACCAATCTGAAGTTC, designated herein as SEQ ID NO: 103). Theprimer probe sets target different regions of the mouse sequence.

Results are presented in Table 12 and are the average of the valuesmeasured in three separate experiments. The results demonstrate blockingof splicing, as represented by EDA⁺FN expression. The expression valueof untreated cells was taken as 1.00. ‘n.d.’ indicates that the mRNAexpression level values were not considered because the oligonucleotidetargeted an amplicon region of the specific primer probe set.

TABLE 12EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in MHT cellsMis- % % Human matches Mouse inhibition inhibition Start with Start ISISwith with SEQ Site human Sequence Site No LTS01050 LTS01052 ID NO 554690 GCAAATTAATGGTAA 49723 511399  0.43 0.44 104 55486 0 CGATCAATGTCTGTT49740 511404 n.d. 0.27 105 55746 1 CTGTGGACTGGATTC 50000 594673  0.61n.d.  85 55756 1 AACGATATACCTGTG 50010 594675  0.28 n.d.  87 55481 0AATGTCTGTTAGGCA 49735 594681 n.d. 0.37  35 55501 0 GCCAGTCCTTTAGGG 49755594694 n.d. 0.46  43 55531 0 TTGATGGAATCGACA 49785 594699 n.d. 0.93  4855561 0 ACTTGCCCCTGTGGG 49815 594704 n.d. 0.94  53 55591 0GGGCTCGAGTAGGTC 49845 594709  0.18 0.37  58 55711 0 ATATCATCGTGCAAG49965 594729  0.50 0.33  78 55418 3 TCCATACCATGCAAA 49670 598110  1.111.04 106 20288 3 ATATTTCCATACCAT 49675 598111  1.11 1.02 107 51489 317531 3 CAAGCATATTTCCAT 49680 598112  0.93 1.03 108 19804 3 23642 342202 3 43710 2 TGAAACAAGCATATT 49685 598113  1.04 1.15 109 55431 255436 1 AGTTGTGAAACAAGC 49690 598114  0.42 0.58 110 55441 0AAGCAAGTTGTGAAA 49695 598115  0.08 0.35 111 55446 0 GTGAAAAGCAAGTTG49700 598116  0.85 0.94 112 55451 0 GTTATGTGAAAAGCA 49705 598117  0.630.68 113 55456 0 AAGAGGTTATGTGAA 49710 598118  0.58 0.54 114 55461 0ATGGTAAGAGGTTAT 49715 598119  0.47 0.43 115 55466 0 AATTAATGGTAAGAG49720 598120  0.49 0.53 116 55471 0 AGGCAAATTAATGGT 49725 598121  0.270.29 117 55476 0 CTGTTAGGCAAATTA 49730 598122  0.23 0.34 118 55491 0TAGGGCGATCAATGT 49745 598123 n.d. 0.29 119 55496 0 TCCTTTAGGGCGATC 49750598124 n.d. 0.39 120 55506 0 TGAATGCCAGTCCTT 49760 598125 n.d. 0.70 12155511 0 ATCAGTGAATGCCAG 49765 598126 n.d. 0.54 122 55516 0TCCACATCAGTGAAT 49770 598127 n.d. 0.53 123 55521 0 CGACATCCACATCAG 49775598128 n.d. 0.47 124 55526 0 GGAATCGACATCCAC 49780 598129 n.d. 0.32 12555536 0 CAATTTTGATGGAAT 49790 598130 n.d. 0.26 126 55541 0CCAAGCAATTTTGAT 49795 598131 n.d. 0.45 127 55546 0 CTTTCCCAAGCAATT 49800598132 n.d. 0.51 128 55551 0 GTGGGCTTTCCCAAG 49805 598133 n.d. 0.92 12955556 0 CCCCTGTGGGCTTTC 49810 598134 n.d. 0.59 130 55566 0TGGAAACTTGCCCCT 49820 598135 n.d. 0.70 131 55571 0 GTACCTGGAAACTTG 49825598136 n.d. 0.50 132 55576 0 ACCCTGTACCTGGAA 49830 598137 78 0.27 13355581 0 AGGTCACCCTGTACC 49835 598138 78 0.27 134 55586 0 CGAGTAGGTCACCCT49840 598139 71 0.41 135 55596 0 CCTCAGGGCTCGAGT 49850 598140 73 0.42136 55601 0 TCCATCCTCAGGGCT 49855 598141 63 0.42 137 55606 1CGGATTCCATCCTCA 49860 598142 52 0.35 138 55611 2 GCTCCCGGATTCCAT 49865598143 78 0.31 139 48384 3 GAAAAGCTCCCGGAT 49870 598144  0.20 0.28 14055616 3 55621 2 GCAGGGAAAAGCTCC 49875 598145  0.19 0.21 141 55626 1CAGGTGCAGGGAAAA 49880 598146  0.32 0.33 142 55631 0 ACCATCAGGTGCAGG49885 598147  0.31 0.30 143 55636 0 TCTTCACCATCAGGT 49890 598148  0.470.38 144 55641 1 TGTCGTCTTCACCAT 49895 598149  0.39 0.37 145 55646 1TGCAGTGTCGTCTTC 49900 598150  0.66 0.41 146 55651 1 AGCTCTGCAGTGTCG49905 598151  0.29 0.24 147 55656 1 CCTGCAGCTCTGCAG 49910 598152  0.400.42 148 55661 1 GAGGCCCTGCAGCTC 49915 598153  0.25 0.21 149 55666 2GGCCTGAGGCCCTGC 49920 598154  0.79 0.80 150 55671 2 ACCCCGGCCTGAGGC49925 598155  0.48 0.54 151 55676 2 CTCAGACCCCGGCCT 49930 598156  0.680.76 152 55681 1 GTGTACTCAGACCCC 49935 598157  0.57 0.41 153 55686 0TGACTGTGTACTCAG 49940 598158  0.56 0.54 154 55691 0 CACACTGACTGTGTA49945 598159  0.59 0.51 155 55696 0 GCAACCACACTGACT 49950 598160  0.540.49 156 55701 0 GCAAGGCAACCACAC 49955 598161  0.27 0.27 157 55706 0ATCGTGCAAGGCAAC 49960 598162  0.56 0.49 158 55716 0 TCTCCATATCATCGT49970 598163  0.31 0.22 159 55721 0 CTGGCTCTCCATATC 49975 598164  0.66n.d. 160 55741 1 GACTGGATTCCAATC 49995 598165  0.56 n.d. 161 55751 0TATACCTGTGGACTG 50005 598166  0.39 n.d. 162 55761 3 CGGTTAACGATATAC50015 598167  0.30 0.25 163 n/a n/a GGGTGCGGTTAACGA 50020 598168  0.910.95 164 n/a n/a GTGGTGGGTGCGGTT 50025 598169  0.95 0.51 165 7620 3CCCGGGTGGTGGGTG 50030 598170  0.81 0.79 166 n/a n/a AAGCACCCGGGTGGT50035 598171  0.92 1.00 167 n/a n/a CCCAGAAGCACCCGG 50040 598172  1.361.32 168 50882 3 CTGTTCCCAGAAGCA 50045 598173  1.44 1.41 169 23948 1AGCCACTGTTCCCAG 50050 598174  1.11 1.14 170 47171 1 55796 2CATAAAGCCACTGTT 50055 598175  0.94 0.91 171 64745 2 55801 3CAAGGCATAAAGCCA 50060 598176  1.13 0.97 172 n/a n/a GCCAGCAAGGCATAA50065 598177  0.78 0.95 173 61226 3 ATAACGCCAGCAAGG 50070 598178  1.081.10 174 n/a n/a AAAGTATAACGCCAG 50075 598179  1.13 1.08 175 3323 3CCAGTAAAGTATAAC 50080 598180  1.09 1.20 176 55824 3 60705 3

Example 9: Antisense Inhibition of Fibronectin mRNA in MHT Cells byUniform MOE Oligonucleotides Designed by Microwalk

Additional antisense oligonucleotides were designed based on the ISISoligonucleotides that demonstrated significant effect on fibronectinsplicing in the studies described above. These oligonucleotides weredesigned by creating oligonucleotides shifted slightly upstream anddownstream (i.e. “microwalk”) of ISIS 511417, ISIS 594685, ISIS 594686,ISIS 594686, ISIS 594687, ISIS 594688, ISIS 594689, ISIS 594690, ISIS594691, and ISIS 598145. The newly designed antisense oligonucleotidesin Tables 13 and 14 were designed as uniform MOE oligonucleotides. Eacholigonucleotide is 18 nucleosides long and each nucleoside in theoligonucleotide has a 2′-MOE modification. The internucleoside linkagesthroughout each oligonucleotide are phosphorothioate (P═S) linkages. Allcytosine residues throughout each oligonucleotide are 5-methylcytosines.The oligonucleotides are presented in the tables below. “Human Startsite” indicates the 5′-most nucleoside to which the oligonucleotide istargeted in the human gene sequence. “Murine Start Site” indicates the5′-most nucleoside to which the oligonucleotide is targeted murine genesequence. Each oligonucleotide listed in the tables is targeted to thehuman fibronectin genomic sequence, SEQ ID NO: 1 or to murinefibronectin genomic sequence, SEQ ID NO: 29, or both. Several of theoligonucleotides are cross-reactive with human and mouse gene sequences.The greater the complementarity between the oligonucleotide and the genesequence, the more likely the oligonucleotide can target the genesequence. ‘Mismatches’ indicates the least number of nucleotides in theoligonucleotide that are mismatched with the gene sequence; theantisense oligonucleotide may target the gene sequence with moremismatches. ‘n/a’ indicates that the antisense oligonucleotide has morethan 3 mismatches with the particular gene sequence.

Cultured MHT cells were transfected using 5 μl LipofectAMINE2000®/mLOptiMEM with 10 nM antisense oligonucleotide. After a treatment periodof approximately 4 hours, the medium was removed and new medium wasadded, left in culture overnight. RNA was isolated from the cells andmeasured by RT-PCR. EDA⁺FN mRNA expression was measured with mouseprimer probe set LTS01050, as well as with LTS01052.

Results are presented in Tables 13 and 14, and are the average of thevalues measured in three separate experiments. The results demonstrateblocking of splicing, as represented by EDA⁺FN expression. Theexpression value of untreated cells was taken as 1.00. ‘n.d.’ indicatesthat the mRNA expression level values were not considered because theoligonucleotide targeted an amplicon region of the specific primer probeset.

TABLE 13EDATN mRNA levels compared to untreated cells (designated 1.00) in MHT cellsMis- matches Human with Murine SEQ Start human ISIS Start ID Sitesequence Sequence No Site LTS01050 LTS01052 NO 55471 0GTTAGGCAAATTAATGGT 606663 49725 n.d. 0.77 177 55472 0 TGTTAGGCAAATTAATGG606664 49726 n.d. 0.80 178 55473 0 CTGTTAGGCAAATTAATG 606665 49727 n.d.0.78 179 55474 0 TCTGTTAGGCAAATTAAT 606666 49728 n.d. 0.73 180 55475 0GTCTGTTAGGCAAATTAA 606667 49729 n.d. 0.70 181 55476 0 TGTCTGTTAGGCAAATTA606668 49730 n.d. 0.62 182 55477 0 ATGTCTGTTAGGCAAATT 606669 49731 n.d.0.66 183 55478 0 AATGTCTGTTAGGCAAAT 594685 49732 n.d. 0.79  32 55479 0CAATGTCTGTTAGGCAAA 594686 49733 0.57 0.50  33 55480 0 TCAATGTCTGTTAGGCAA594687 49734 1.21 0.99  34 55481 0 ATCAATGTCTGTTAGGCA 594688 49735 1.040.83  36 55482 0 GATCAATGTCTGTTAGGC 511417 49736 0.70 0.63 184 55483 0CGATCAATGTCTGTTAGG 594689 49737 n.d. 0.66  38 55484 0 GCGATCAATGTCTGTTAG594690 49738 n.d. 0.64  40 55485 0 GGCGATCAATGTCTGTTA 594691 49739 n.d.0.85  42 55486 0 GGGCGATCAATGTCTGTT 606670 49740 n.d. 0.83 185 55487 0AGGGCGATCAATGTCTGT 606671 49741 n.d. 0.82 186 55488 0 TAGGGCGATCAATGTCTG606672 49742 n.d. 0.80 187 55489 0 TTAGGGCGATCAATGTCT 606673 49743 n.d.0.56 188 55490 0 TTTAGGGCGATCAATGTC 606674 49744 n.d. 0.56 189 55491 0CTTTAGGGCGATCAATGT 606675 49745 n.d. 0.55 190 55492 0 CCTTTAGGGCGATCAATG606676 49746 n.d. 0.47 191 55493 0 TCCTTTAGGGCGATCAAT 606677 49747 n.d.0.57 192 55494 0 GTCCTTTAGGGCGATCAA 606678 49748 n.d. 0.62 193 55495 0AGTCCTTTAGGGCGATCA 606679 49749 n.d. 0.94 194 55496 0 CAGTCCTTTAGGGCGATC606680 49750 n.d. 0.92 195 55523 0 GGAATCGACATCCACATC 606681 49777 n.d.0.68 196 55524 0 TGGAATCGACATCCACAT 606682 49778 n.d. 0.70 197 55525 0ATGGAATCGACATCCACA 606683 49779 n.d. 0.66 198 55526 0 GATGGAATCGACATCCAC606684 49780 n.d. 0.63 199 55527 0 TGATGGAATCGACATCCA 606685 49781 n.d.0.63 200 55528 0 TTGATGGAATCGACATCC 606686 49782 n.d. 0.88 201 55529 0TTTGATGGAATCGACATC 606687 49783 n.d. 0.93 202 55533 0 CAATTTTGATGGAATCGA606688 49787 n.d. 1.05 203 55534 0 GCAATTTTGATGGAATCG 606689 49788 n.d.0.81 204 55535 0 AGCAATTTTGATGGAATC 606690 49789 n.d. 0.60 205 55536 0AAGCAATTTTGATGGAAT 606691 49790 n.d. 0.66 206 55537 0 CAAGCAATTTTGATGGAA606692 49791 n.d. 0.56 207 55538 0 CCAAGCAATTTTGATGGA 606693 49792 n.d.0.61 208 55539 0 CCCAAGCAATTTTGATGG 606694 49793 n.d. 0.84 209 55576 0GTCACCCTGTACCTGGAA 606695 49830 n.d. 0.88 210 55577 0 GGTCACCCTGTACCTGGA606696 49831 1.32 1.07 211 55578 0 AGGTCACCCTGTACCTGG 606697 49832 0.740.81 212 55579 0 TAGGTCACCCTGTACCTG 606698 49833 0.61 0.69 213 55580 0GTAGGTCACCCTGTACCT 606699 49834 0.52 0.49 214 55581 0 AGTAGGTCACCCTGTACC606700 49835 0.48 0.57 215 55582 0 GAGTAGGTCACCCTGTAC 606701 49836 0.450.59 216 55583 0 CGAGTAGGTCACCCTGTA 606702 49837 0.60 0.80 217 55584 0TCGAGTAGGTCACCCTGT 606703 49838 0.60 0.91 218 55585 0 CTCGAGTAGGTCACCCTG606704 49839 0.60 0.78 219 55586 0 GCTCGAGTAGGTCACCCT 606705 49840 0.590.69 220 55587 0 GGCTCGAGTAGGTCACCC 606706 49841 0.57 0.57 221 55588 0GGGCTCGAGTAGGTCACC 606707 49842 0.53 0.58 222 55589 0 AGGGCTCGAGTAGGTCAC606708 49843 0.62 0.63 223 55590 0 CAGGGCTCGAGTAGGTCA 606709 49844 0.450.50 224 55591 0 TCAGGGCTCGAGTAGGTC 606710 49845 0.60 0.60 225 55592 0CTCAGGGCTCGAGTAGGT 606711 49846 0.83 0.86 226 55593 0 CCTCAGGGCTCGAGTAGG606712 49847 0.95 0.99 227 55594 0 TCCTCAGGGCTCGAGTAG 606713 49848 0.950.83 228 55595 0 ATCCTCAGGGCTCGAGTA 606714 49849 0.67 0.61 229 55596 0CATCCTCAGGGCTCGAGT 606715 49850 0.58 0.59 230 55597 0 CCATCCTCAGGGCTCGAG606716 49851 0.65 0.58 231 55598 0 TCCATCCTCAGGGCTCGA 606717 49852 0.640.54 232 55599 0 TTCCATCCTCAGGGCTCG 606718 49853 0.65 0.62 233 55600 0ATTCCATCCTCAGGGCTC 606719 49854 0.87 0.88 234 55601 0 GATTCCATCCTCAGGGCT606720 49855 0.87 0.82 235 55602 0 GGATTCCATCCTCAGGGC 606721 49856 0.530.66 236 55603 1 CGGATTCCATCCTCAGGG 606722 49857 0.48 0.51 237 55604 2CCGGATTCCATCCTCAGG 606723 49858 0.58 0.50 238 55605 2 CCCGGATTCCATCCTCAG606724 49859 0.53 0.49 239 55606 2 TCCCGGATTCCATCCTCA 606725 49860 0.510.53 240 55607 2 CTCCCGGATTCCATCCTC 606726 49861 0.61 0.62 241 55608 2GCTCCCGGATTCCATCCT 606727 49862 0.68 0.75 242 55609 2 AGCTCCCGGATTCCATCC606728 49863 0.83 0.90 243 55610 3 AAGCTCCCGGATTCCATC 606729 49864 0.480.68 244 55611 3 AAAGCTCCCGGATTCCAT 606730 49865 0.26 0.53 245 55612 3AAAAGCTCCCGGATTCCA 606731 49866 0.32 0.52 246 55613 3 GAAAAGCTCCCGGATTCC606732 49867 0.37 0.52 247 55614 3 GGAAAAGCTCCCGGATTC 606733 49868 0.490.59 248 55615 3 GGGAAAAGCTCCCGGATT 606734 49869 0.61 0.57 249 55621 2GCAGGGAAAAGCTCC 598145 49875 0.78 0.92 141

TABLE 14EDATN mRNA levels compared to untreated cells (designated 1.00) in MHT cellsMis- matches Human with Murine SEQ Start human ISIS Start ID Sitesequence Sequence No Site LTS01050 LTS01052 NO 48384 3AGGGAAAAGCTCCCGGAT 606735 49870 0.95 0.77 250 55617 3 CAGGGAAAAGCTCCCGGA606736 49871 1.04 0.83 251 55618 3 GCAGGGAAAAGCTCCCGG 606737 49872 0.850.82 252 55619 3 TGCAGGGAAAAGCTCCCG 606738 49873 0.71 0.76 253 55620 3GTGCAGGGAAAAGCTCCC 606739 49874 0.65 0.68 254 55621 2 GCAGGGAAAAGCTCC598145 49875 0.79 0.55 141 55621 2 GGTGCAGGGAAAAGCTCC 606740 49875 0.790.76 255 55622 1 AGGTGCAGGGAAAAGCTC 606741 49876 0.88 0.83 256 55623 1CAGGTGCAGGGAAAAGCT 606742 49877 0.77 0.88 257 55624 1 TCAGGTGCAGGGAAAAGC606743 49878 1.11 0.98 258 55625 1 ATCAGGTGCAGGGAAAAG 606744 49879 1.050.97 259 55626 1 CATCAGGTGCAGGGAAAA 606745 49880 0.76 0.79 260 55627 1CCATCAGGTGCAGGGAAA 606746 49881 0.50 0.63 261 55628 0 ACCATCAGGTGCAGGGAA606747 49882 0.53 0.73 262 55629 0 CACCATCAGGTGCAGGGA 606748 49883 0.700.77 263 55630 0 TCACCATCAGGTGCAGGG 606749 49884 0.83 0.85 264 55631 0TTCACCATCAGGTGCAGG 606750 49885 1.11 0.94 265 55632 0 CTTCACCATCAGGTGCAG606751 49886 0.88 0.82 266 55633 0 TCTTCACCATCAGGTGCA 606752 49887 0.680.71 267 55634 1 GTCTTCACCATCAGGTGC 606753 49888 0.49 0.55 268 55635 1CGTCTTCACCATCAGGTG 606754 49889 0.90 0.53 269 55636 1 TCGTCTTCACCATCAGGT606755 49890 1.17 0.49 270 55637 1 GTCGTCTTCACCATCAGG 606756 49891 1.580.57 271 55638 1 TGTCGTCTTCACCATCAG 606757 49892 1.48 0.64 272 55639 1GTGTCGTCTTCACCATCA 606758 49893 1.27 0.96 273 55640 1 AGTGTCGTCTTCACCATC606759 49894 0.78 0.75 274 55641 1 CAGTGTCGTCTTCACCAT 606760 49895 0.520.54 275 55642 1 GCAGTGTCGTCTTCACCA 606761 49896 0.47 0.53 276 55643 1TGCAGTGTCGTCTTCACC 606762 49897 0.49 0.55 277 55644 1 CTGCAGTGTCGTCTTCAC606763 49898 0.61 0.54 278 55645 1 TCTGCAGTGTCGTCTTCA 606764 49899 0.930.57 279 55646 1 CTCTGCAGTGTCGTCTTC 606765 49900 0.98 0.67 280 55647 1GCTCTGCAGTGTCGTCTT 606766 49901 0.72 0.83 281 55648 1 AGCTCTGCAGTGTCGTCT606767 49902 0.71 0.65 282 55649 1 CAGCTCTGCAGTGTCGTC 606768 49903 0.710.62 283 55650 1 GCAGCTCTGCAGTGTCGT 606769 49904 0.64 0.60 284 55651 1TGCAGCTCTGCAGTGTCG 606770 49905 0.40 0.47 285 55652 1 CTGCAGCTCTGCAGTGTC606771 49906 0.35 0.53 286 55653 1 CCTGCAGCTCTGCAGTGT 606772 49907 0.470.56 287 55654 1 CCCTGCAGCTCTGCAGTG 606773 49908 0.71 0.97 288 55655 1GCCCTGCAGCTCTGCAGT 606774 49909 0.83 0.97 289 55656 1 GGCCCTGCAGCTCTGCAG606775 49910 0.91 0.73 290 55657 1 AGGCCCTGCAGCTCTGCA 606776 49911 0.740.64 291 55658 1 GAGGCCCTGCAGCTCTGC 606777 49912 0.60 0.66 292 55659 1TGAGGCCCTGCAGCTCTG 606778 49913 0.49 0.50 293 55660 1 CTGAGGCCCTGCAGCTCT606779 49914 0.47 0.57 294 55661 2 CCTGAGGCCCTGCAGCTC 606780 49915 0.540.65 295 55698 0 GCAAGGCAACCACACTGA 606781 49952 0.60 0.71 296 55699 0TGCAAGGCAACCACACTG 606782 49953 0.75 0.79 297 55700 0 GTGCAAGGCAACCACACT606783 49954 0.75 0.63 298 55701 0 CGTGCAAGGCAACCACAC 606784 49955 0.530.59 299 55702 0 TCGTGCAAGGCAACCACA 606785 49956 0.46 0.46 300 55703 0ATCGTGCAAGGCAACCAC 606786 49957 0.48 0.55 301 55704 0 CATCGTGCAAGGCAACCA606787 49958 0.46 0.45 302 55708 0 ATATCATCGTGCAAGGCA 606788 49962 0.360.45 303 55709 0 CATATCATCGTGCAAGGC 606789 49963 0.59 0.49 304 55710 0CCATATCATCGTGCAAGG 606790 49964 1.00 0.72 305 55711 0 TCCATATCATCGTGCAAG606791 49965 1.00 0.58 306 55712 0 CTCCATATCATCGTGCAA 606792 49966 0.780.54 307 55713 0 TCTCCATATCATCGTGCA 606793 49967 0.61 0.46 308 55714 0CTCTCCATATCATCGTGC 606794 49968 0.52 n.d. 309 55715 0 GCTCTCCATATCATCGTG606795 49969 0.69 n.d. 310 55716 0 GGCTCTCCATATCATCGT 606796 49970 0.70n.d. 311 55717 0 TGGCTCTCCATATCATCG 606797 49971 0.71 n.d. 312 55718 0CTGGCTCTCCATATCATC 606798 49972 1.37 n.d. 313 55719 0 GCTGGCTCTCCATATCAT606799 49973 1.82 n.d. 314 55749 1 ATATACCTGTGGACTGGA 606800 50003 0.77n.d. 315 55750 1 GATATACCTGTGGACTGG 606801 50004 0.56 n.d. 316 55751 1CGATATACCTGTGGACTG 606802 50005 0.40 n.d. 317 55752 1 ACGATATACCTGTGGACT606803 50006 0.34 n.d. 318 55753 1 AACGATATACCTGTGGAC 606804 50007 0.33n.d. 319 55754 1 TAACGATATACCTGTGGA 606805 50008 0.47 n.d. 320 55755 1TTAACGATATACCTGTGG 606806 50009 0.57 n.d. 321 55756 2 GTTAACGATATACCTGTG606807 50010 0.73 n.d. 322 55757 3 GGTTAACGATATACCTGT 606808 50011 0.59n.d. 323 55758 3 CGGTTAACGATATACCTG 606809 50012 0.40 n.d. 324 55759 3GCGGTTAACGATATACCT 606810 50013 0.40 n.d. 325 55760 3 TGCGGTTAACGATATACC606811 50014 0.40 n.d. 326 55761 3 GTGCGGTTAACGATATAC 606812 50015 0.430.46 327 55762 3 GGTGCGGTTAACGATATA 606813 50016 0.48 0.59 328 n/a n/aGGGTGCGGTTAACGATAT 606814 50017 0.77 0.83 329

Example 10: In Vitro Screening of Antisense Oligonucleotides with(S)-cEt Modifications Targeting Human and/or Mouse Fibronectin in b.ENDCells

Antisense oligonucleotides were designed targeting a fibronectin nucleicacid and were tested for their effects on blocking of fibronectinsplicing in vitro. ISIS 606793 was also included in the study. The newlydesigned antisense oligonucleotides in Tables 15-22 were designed asdeoxy and (S)-cEt oligonucleotides. Each nucleoside in theoligonucleotide has a 2′-MOE, deoxy, or (S)-cEt modification, aspresented in the Chemistry column of the tables. ‘e’ indicates MOE; ‘k’indicates (S)-cEt; ‘d’ indicates deoxy modifications. Theinternucleoside linkages throughout each oligonucleotide arephosphorothioate (P═S) linkages. All cytosine residues throughout eacholigonucleotide are 5-methylcytosines. “Human Start site” indicates the5′-most nucleoside to which the oligonucleotide is targeted in the humangene sequence. “Murine Start Site” indicates the 5′-most nucleoside towhich the oligonucleotide is targeted murine gene sequence. Eacholigonucleotide listed in Tables 15-22 is targeted to the humanfibronectin genomic sequence, SEQ ID NO: 1 or to murine fibronectingenomic sequence, SEQ ID NO: 29, or both. Several of theoligonucleotides are cross-reactive with human and mouse gene sequences.The greater the complementarity between the oligonucleotide and the genesequence, the more likely the oligonucleotide can target the genesequence. ‘Mismatches’ indicates the least number of nucleotides in theoligonucleotide that are mismatched with the gene sequence; theantisense oligonucleotide may target the gene sequence with moremismatches. ‘n/a’ indicates that the antisense oligonucleotide has morethan 3 mismatches with the particular gene sequence.

Cultured b.END cells were transfected using 2 μl Cytofectin/mL with 3 nMantisense oligonucleotide. After a treatment period of approximately 4hours, the medium was removed and new medium was added, left in cultureovernight. RNA was isolated from the cells and measured by RT-PCR.EDA⁺FN mRNA expression was measured with mouse primer probe setLTS01050, as well as with LTS01052.

Results are presented in Tables 15-22, and are the average of the valuesmeasured in three separate experiments. The results demonstrate blockingof splicing, as represented by EDA⁺FN expression. The expression valueof untreated cells was taken as 1.00. ‘n.d.’ indicates that the mRNAexpression level values were not considered because the oligonucleotidetargeted an amplicon region of the specific primer probe set.

TABLE 15EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in b.END cellsmeasured with LTS01050 Mis- matches Human to the Murine SEQ Start humanStart ISIS EDA + ID Site sequence Site Sequence No Chemistry FN NO 483863 49872 AGGGAAAAGCTCCCGG 607180 kddkddkddkddkddk 0.80 330 55578 0 49832GTCACCCTGTACCTGG 607170 kddkddkddkddkddk 0.86 331 55582 0 49836GTAGGTCACCCTGTAC 607171 kddkddkddkddkddk 0.71 332 55586 0 49840TCGAGTAGGTCACCCT 607172 kddkddkddkddkddk 0.56 333 55590 0 49844GGGCTCGAGTAGGTCA 607173 kddkddkddkddkddk 0.41 334 55594 0 49848CTCAGGGCTCGAGTAG 607174 kddkddkddkddkddk 0.50 335 55598 0 49852CATCCTCAGGGCTCGA 607175 kddkddkddkddkddk 0.44 336 55602 0 49856ATTCCATCCTCAGGGC 607176 kddkddkddkddkddk 0.42 337 55606 2 49860CCGGATTCCATCCTCA 607177 kddkddkddkddkddk 0.47 338 55610 2 49864GCTCCCGGATTCCATC 607178 kddkddkddkddkddk 0.18 339 55614 3 49868AAAAGCTCCCGGATTC 607179 kddkddkddkddkddk 0.31 340 55622 1 49876GTGCAGGGAAAAGCTC 607181 kddkddkddkddkddk 0.73 341 55626 1 49880TCAGGTGCAGGGAAAA 607182 kddkddkddkddkddk 0.72 342 55630 0 49884ACCATCAGGTGCAGGG 607183 kddkddkddkddkddk 0.36 343 55634 0 49888CTTCACCATCAGGTGC 607184 kddkddkddkddkddk 0.49 344 55638 1 49892TCGTCTTCACCATCAG 607185 kddkddkddkddkddk 0.34 345 55642 1 49896AGTGTCGTCTTCACCA 607186 kddkddkddkddkddk 0.22 346 55646 1 49900CTGCAGTGTCGTCTTC 607187 kddkddkddkddkddk 0.36 347 55650 1 49904AGCTCTGCAGTGTCGT 607188 kddkddkddkddkddk 0.19 348 55654 1 49908CTGCAGCTCTGCAGTG 607189 kddkddkddkddkddk 0.71 349 55658 1 49912GGCCCTGCAGCTCTGC 607190 kddkddkddkddkddk 0.29 350 55662 1 49916CTGAGGCCCTGCAGCT 607191 kddkddkddkddkddk 0.32 351 55666 2 49920CGGCCTGAGGCCCTGC 607192 kddkddkddkddkddk 0.48 352 55670 2 49924ACCCCGGCCTGAGGCC 607193 kddkddkddkddkddk 0.33 353 55674 2 49928TCAGACCCCGGCCTGA 607194 kddkddkddkddkddk 0.34 354 55678 2 49932GTACTCAGACCCCGGC 607195 kddkddkddkddkddk 0.27 355 55682 1 49936CTGTGTACTCAGACCC 607196 kddkddkddkddkddk 0.63 356 55686 0 49940CTGACTGTGTACTCAG 607197 kddkddkddkddkddk 0.61 357 55690 0 49944CACACTGACTGTGTAC 607198 kddkddkddkddkddk 0.57 358 55694 0 49948CAACCACACTGACTGT 607199 kddkddkddkddkddk 0.51 359 55698 0 49952AAGGCAACCACACTGA 607200 kddkddkddkddkddk 0.47 360 55702 0 49956GTGCAAGGCAACCACA 607201 kddkddkddkddkddk 0.28 361 55706 0 49960CATCGTGCAAGGCAAC 607202 kddkddkddkddkddk 0.27 362 55710 0 49964ATATCATCGTGCAAGG 607203 kddkddkddkddkddk 0.44 363 55713 0 49967TCTCCATATCATCGTGCA 606793 eeeeeeeeeeeeeeeeee 0.13 308 55714 0 49968CTCCATATCATCGTGC 607204 kddkddkddkddkddk 0.18 364 55718 0 49972GGCTCTCCATATCATC 607205 kddkddkddkddkddk 0.22 365 55722 0 49976GGCTGGCTCTCCATAT 607206 kddkddkddkddkddk 0.53 366 55738 1 49992CTGGATTCCAATCAGG 607207 kddkddkddkddkddk 0.17 367 55742 1 49996TGGACTGGATTCCAAT 607208 kddkddkddkddkddk 0.17 368 55746 1 50000CCTGTGGACTGGATTC 607209 kddkddkddkddkddk 0.23 369 55750 0 50004TATACCTGTGGACTGG 607210 kddkddkddkddkddk 0.24 370 55754 1 50008ACGATATACCTGTGGA 607211 kddkddkddkddkddk 0.51 371 55758 2 50012GTTAACGATATACCTG 607212 kddkddkddkddkddk 0.40 372 55762 3 50016TGCGGTTAACGATATA 607213 kddkddkddkddkddk 0.18 373 n/a n/a 50020TGGGTGCGGTTAACGA 607214 kddkddkddkddkddk 1.39 374

TABLE 16EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in b.END cellsmeasured with LTS01052 Mis- matches Human to the Murine SEQ Start humanStart ISIS EDA + ID Site sequence Site Sequence No Chemistry FN NO 554660 49720 AAATTAATGGTAAGAG 607142 kddkddkddkddkddk 0.19 375 55470 0 49724AGGCAAATTAATGGTA 607143 kddkddkddkddkddk 0.20 376 55474 0 49728TGTTAGGCAAATTAAT 607144 kddkddkddkddkddk 0.53 377 55478 0 49732TGTCTGTTAGGCAAAT 607145 kddkddkddkddkddk 0.59 378 55482 0 49736TCAATGTCTGTTAGGC 607146 kddkddkddkddkddk 0.59 379 55486 0 49740GCGATCAATGTCTGTT 607147 kddkddkddkddkddk 0.65 380 55490 0 49744TAGGGCGATCAATGTC 607148 kddkddkddkddkddk 0.67 381 55494 0 49748CCTTTAGGGCGATCAA 607149 kddkddkddkddkddk 0.68 382 55498 0 49752CAGTCCTTTAGGGCGA 607150 kddkddkddkddkddk 0.68 383 55502 0 49756ATGCCAGTCCTTTAGG 607151 kddkddkddkddkddk 0.71 384 55506 0 49760GTGAATGCCAGTCCTT 607152 kddkddkddkddkddk 0.75 385 55510 0 49764ATCAGTGAATGCCAGT 607153 kddkddkddkddkddk 0.75 386 55514 0 49768CCACATCAGTGAATGC 607154 kddkddkddkddkddk 0.83 387 55518 0 49772ACATCCACATCAGTGA 607155 kddkddkddkddkddk 0.84 388 55522 0 49776ATCGACATCCACATCA 607156 kddkddkddkddkddk 0.88 389 55526 0 49780TGGAATCGACATCCAC 607157 kddkddkddkddkddk 0.90 390 55530 0 49784TTGATGGAATCGACAT 607158 kddkddkddkddkddk 0.91 391 55534 0 49788AATTTTGATGGAATCG 607159 kddkddkddkddkddk 0.91 392 55538 0 49792AAGCAATTTTGATGGA 607160 kddkddkddkddkddk 0.91 393 55542 0 49796TCCCAAGCAATTTTGA 607161 kddkddkddkddkddk 0.95 394 55546 0 49800GCTTTCCCAAGCAATT 607162 kddkddkddkddkddk 0.96 395 55550 0 49804GTGGGCTTTCCCAAGC 607163 kddkddkddkddkddk 0.97 396 55554 0 49808CCCTGTGGGCTTTCCC 607164 kddkddkddkddkddk 0.97 397 55558 0 49812TTGCCCCTGTGGGCTT 607165 kddkddkddkddkddk 1.00 398 55562 0 49816AAACTTGCCCCTGTGG 607166 kddkddkddkddkddk 1.06 399 55566 0 49820CTGGAAACTTGCCCCT 607167 kddkddkddkddkddk 1.09 400 55570 0 49824GTACCTGGAAACTTGC 607168 kddkddkddkddkddk 1.16 401 55574 0 49828CCCTGTACCTGGAAAC 607169 kddkddkddkddkddk 1.17 402 55713 0 49967TCTCCATATCATCGTGCA 606793 eeeeeeeeeeeeeeeeee 0.16 308

TABLE 17EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in b.END cellsmeasured with LTS01050 Mis- matches Human to the Murine SEQ Start humanStart ISIS EDA + ID Site sequence Site Sequence No Chemistry FN NO 557130 49967 TCTCCATATCATCGTGCA 606793 eeeeeeeeeeeeeeeeee 0.16 308 55578 049832 GTCACCCTGTACCTGG 607243 keekeekeekeekeek 0.83 331 55582 0 49836GTAGGTCACCCTGTAC 607244 keekeekeekeekeek 0.97 332 55586 0 49840TCGAGTAGGTCACCCT 607245 keekeekeekeekeek 0.27 333 55590 0 49844GGGCTCGAGTAGGTCA 607246 keekeekeekeekeek 0.65 334 55594 0 49848CTCAGGGCTCGAGTAG 607247 keekeekeekeekeek 0.71 335 55598 0 49852CATCCTCAGGGCTCGA 607248 keekeekeekeekeek 0.85 336 55602 0 49856ATTCCATCCTCAGGGC 607249 keekeekeekeekeek 0.59 337 55606 2 49860CCGGATTCCATCCTCA 607250 keekeekeekeekeek 0.72 338 55610 2 49864GCTCCCGGATTCCATC 607251 keekeekeekeekeek 0.52 339 55614 3 49868AAAAGCTCCCGGATTC 607252 keekeekeekeekeek 0.38 340 48386 3 49872AGGGAAAAGCTCCCGG 607253 keekeekeekeekeek 0.64 330 55622 1 49876GTGCAGGGAAAAGCTC 607254 keekeekeekeekeek 0.61 341 55626 1 49880TCAGGTGCAGGGAAAA 607255 keekeekeekeekeek 0.80 342 55630 0 49884ACCATCAGGTGCAGGG 607256 keekeekeekeekeek 0.47 343 55634 0 49888CTTCACCATCAGGTGC 607257 keekeekeekeekeek 0.83 344 55638 1 49892TCGTCTTCACCATCAG 607258 keekeekeekeekeek 0.53 345 55642 1 49896AGTGTCGTCTTCACCA 607259 keekeekeekeekeek 0.40 346 55646 1 49900CTGCAGTGTCGTCTTC 607260 keekeekeekeekeek 0.84 347 55650 1 49904AGCTCTGCAGTGTCGT 607261 keekeekeekeekeek 0.55 348 55654 1 49908CTGCAGCTCTGCAGTG 607262 keekeekeekeekeek 0.93 349 55658 1 49912GGCCCTGCAGCTCTGC 607263 keekeekeekeekeek 0.88 350 55662 1 49916CTGAGGCCCTGCAGCT 607264 keekeekeekeekeek 0.87 351 55666 2 49920CGGCCTGAGGCCCTGC 607265 keekeekeekeekeek 0.82 352 55670 2 49924ACCCCGGCCTGAGGCC 607266 keekeekeekeekeek 0.46 353 55674 2 49928TCAGACCCCGGCCTGA 607267 keekeekeekeekeek 0.84 354 55678 2 49932GTACTCAGACCCCGGC 607268 keekeekeekeekeek 0.67 355 55682 1 49936CTGTGTACTCAGACCC 607269 keekeekeekeekeek 0.78 356 55686 0 49940CTGACTGTGTACTCAG 607270 keekeekeekeekeek 0.75 357 55690 0 49944CACACTGACTGTGTAC 607271 keekeekeekeekeek 0.90 358 55694 0 49948CAACCACACTGACTGT 607272 keekeekeekeekeek 0.41 359 55698 0 49952AAGGCAACCACACTGA 607273 keekeekeekeekeek 0.30 360 55702 0 49956GTGCAAGGCAACCACA 607274 keekeekeekeekeek 0.35 361 55706 0 49960CATCGTGCAAGGCAAC 607275 keekeekeekeekeek 0.24 362 55710 0 49964ATATCATCGTGCAAGG 607276 keekeekeekeekeek 0.24 363 55714 0 49968CTCCATATCATCGTGC 607277 keekeekeekeekeek 0.20 364 55718 0 49972GGCTCTCCATATCATC 607278 keekeekeekeekeek 0.35 365 55722 0 49976GGCTGGCTCTCCATAT 607279 keekeekeekeekeek 0.84 366 55738 1 49992CTGGATTCCAATCAGG 607280 keekeekeekeekeek 0.28 367 55742 1 49996TGGACTGGATTCCAAT 607281 keekeekeekeekeek 0.54 368 55746 1 50000CCTGTGGACTGGATTC 607282 keekeekeekeekeek 0.42 369 55750 0 50004TATACCTGTGGACTGG 607283 keekeekeekeekeek 0.50 370 55754 1 50008ACGATATACCTGTGGA 607284 keekeekeekeekeek 0.16 371 55758 2 50012GTTAACGATATACCTG 607285 keekeekeekeekeek 0.18 372 55762 3 50016TGCGGTTAACGATATA 607286 keekeekeekeekeek 0.20 373 n/a n/a 50020TGGGTGCGGTTAACGA 607287 keekeekeekeekeek 1.28 374

TABLE 18EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in b.END cellsmeasured with LTS01052 Mis- matches Human to the Murine SEQ Start humanStart ISIS EDA + ID Site sequence Site Sequence No Chemistry FN NO 557130 49967 TCTCCATATCATCGTGCA 606793 eeeeeeeeeeeeeeeeee 0.19 308 55466 049720 AAATTAATGGTAAGAG 607215 keekeekeekeekeek 0.55 375 55470 0 49724AGGCAAATTAATGGTA 607216 keekeekeekeekeek 0.39 376 55474 0 49728TGTTAGGCAAATTAAT 607217 keekeekeekeekeek 0.67 377 55478 0 49732TGTCTGTTAGGCAAAT 607218 keekeekeekeekeek 0.87 378 55482 0 49736TCAATGTCTGTTAGGC 607219 keekeekeekeekeek 0.74 379 55486 0 49740GCGATCAATGTCTGTT 607220 keekeekeekeekeek 0.60 380 55490 0 49744TAGGGCGATCAATGTC 607221 keekeekeekeekeek 0.59 381 55494 0 49748CCTTTAGGGCGATCAA 607222 keekeekeekeekeek 0.71 382 55498 0 49752CAGTCCTTTAGGGCGA 607223 keekeekeekeekeek 0.97 383 55502 0 49756ATGCCAGTCCTTTAGG 607224 keekeekeekeekeek 0.83 384 55506 0 49760GTGAATGCCAGTCCTT 607225 keekeekeekeekeek 1.00 385 55510 0 49764ATCAGTGAATGCCAGT 607226 keekeekeekeekeek 1.09 386 55514 0 49768CCACATCAGTGAATGC 607227 keekeekeekeekeek 0.84 387 55518 0 49772ACATCCACATCAGTGA 607228 keekeekeekeekeek 0.96 388 55522 0 49776ATCGACATCCACATCA 607229 keekeekeekeekeek 0.84 389 55526 0 49780TGGAATCGACATCCAC 607230 keekeekeekeekeek 0.95 390 55530 0 49784TTGATGGAATCGACAT 607231 keekeekeekeekeek 0.96 391 55534 0 49788AATTTTGATGGAATCG 607232 keekeekeekeekeek 0.83 392 55538 0 49792AAGCAATTTTGATGGA 607233 keekeekeekeekeek 0.65 393 55542 0 49796TCCCAAGCAATTTTGA 607234 keekeekeekeekeek 0.73 394 55546 0 49800GCTTTCCCAAGCAATT 607235 keekeekeekeekeek 0.96 395 55550 0 49804GTGGGCTTTCCCAAGC 607236 keekeekeekeekeek 0.93 396 55554 0 49808CCCTGTGGGCTTTCCC 607237 keekeekeekeekeek 0.99 397 55558 0 49812TTGCCCCTGTGGGCTT 607238 keekeekeekeekeek 0.92 398 55562 0 49816AAACTTGCCCCTGTGG 607239 keekeekeekeekeek 0.95 399 55566 0 49820CTGGAAACTTGCCCCT 607240 keekeekeekeekeek 0.79 400 55570 0 49824GTACCTGGAAACTTGC 607241 keekeekeekeekeek 0.68 401 55574 0 49828CCCTGTACCTGGAAAC 607242 keekeekeekeekeek 0.84 402

TABLE 19EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in b.END cellsmeasured with LTS01050 Mis- matches Human to the Murine SEQ Start humanStart ISIS EDA + ID Site sequence Site Sequence No Chemistry FN NO 557130 49967 TCTCCATATCATCGTGCA 606793 eeeeeeeeeeeeeeeeee 0.20 308 55578 049832 AGGTCACCCTGTACCTGG 607388 kkeekeekeekeekeeke 0.76 212 55582 049836 GAGTAGGTCACCCTGTAC 607389 kkeekeekeekeekeeke 0.76 216 55586 049840 GCTCGAGTAGGTCACCCT 607390 kkeekeekeekeekeeke 0.83 220 55590 049844 CAGGGCTCGAGTAGGTCA 607391 kkeekeekeekeekeeke 1.03 224 55594 049848 TCCTCAGGGCTCGAGTAG 607392 kkeekeekeekeekeeke 0.88 228 55598 049852 TCCATCCTCAGGGCTCGA 607393 kkeekeekeekeekeeke 0.74 232 55602 049856 GGATTCCATCCTCAGGGC 607394 kkeekeekeekeekeeke 1.16 236 55606 249860 TCCCGGATTCCATCCTCA 607395 kkeekeekeekeekeeke 0.81 240 55610 349864 AAGCTCCCGGATTCCATC 607396 kkeekeekeekeekeeke 0.71 244 55614 349868 GGAAAAGCTCCCGGATTC 607397 kkeekeekeekeekeeke 0.74 248 55618 349872 GCAGGGAAAAGCTCCCGG 607398 kkeekeekeekeekeeke 0.69 252 55622 149876 AGGTGCAGGGAAAAGCTC 607399 kkeekeekeekeekeeke 0.57 256 55626 149880 CATCAGGTGCAGGGAAAA 607400 kkeekeekeekeekeeke 0.80 260 55630 049884 TCACCATCAGGTGCAGGG 607401 kkeekeekeekeekeeke 0.64 264 55634 149888 GTCTTCACCATCAGGTGC 607402 kkeekeekeekeekeeke 0.65 268 55638 149892 TGTCGTCTTCACCATCAG 607403 kkeekeekeekeekeeke 0.46 272 55642 149896 GCAGTGTCGTCTTCACCA 607404 kkeekeekeekeekeeke 0.51 276 55646 149900 CTCTGCAGTGTCGTCTTC 607405 kkeekeekeekeekeeke 0.39 280 55650 149904 GCAGCTCTGCAGTGTCGT 607406 kkeekeekeekeekeeke 0.75 284 55654 149908 CCCTGCAGCTCTGCAGTG 607407 kkeekeekeekeekeeke 0.40 288 55658 149912 GAGGCCCTGCAGCTCTGC 607408 kkeekeekeekeekeeke 1.00 292 55662 249916 GCCTGAGGCCCTGCAGCT 607409 kkeekeekeekeekeeke 0.64 403 55666 249920 CCCGGCCTGAGGCCCTGC 607410 kkeekeekeekeekeeke 0.24 404 55670 249924 AGACCCCGGCCTGAGGCC 607411 kkeekeekeekeekeeke 0.47 405 55674 249928 ACTCAGACCCCGGCCTGA 607412 kkeekeekeekeekeeke 0.62 406 55678 249932 GTGTACTCAGACCCCGGC 607413 kkeekeekeekeekeeke 0.74 407 55682 149936 GACTGTGTACTCAGACCC 607414 kkeekeekeekeekeeke 0.53 408 55686 049940 CACTGACTGTGTACTCAG 607415 kkeekeekeekeekeeke 1.03 409 55690 049944 ACCACACTGACTGTGTAC 607416 kkeekeekeekeekeeke 0.95 410 55694 049948 GGCAACCACACTGACTGT 607417 kkeekeekeekeekeeke 0.83 411 55698 049952 GCAAGGCAACCACACTGA 607418 kkeekeekeekeekeeke 0.80 296 55702 049956 TCGTGCAAGGCAACCACA 607419 kkeekeekeekeekeeke 0.78 300 55706 049960 ATCATCGTGCAAGGCAAC 607420 kkeekeekeekeekeeke 0.88 412 55710 049964 CCATATCATCGTGCAAGG 607421 kkeekeekeekeekeeke 0.71 305 55714 049968 CTCTCCATATCATCGTGC 607422 kkeekeekeekeekeeke 0.84 309 55718 049972 CTGGCTCTCCATATCATC 607423 kkeekeekeekeekeeke 0.42 313 55738 149992 GACTGGATTCCAATCAGG 607424 kkeekeekeekeekeeke 0.47 413 55742 149996 TGTGGACTGGATTCCAAT 607425 kkeekeekeekeekeeke 0.49 414 55746 150000 TACCTGTGGACTGGATTC 607426 kkeekeekeekeekeeke 0.48 415 55750 150004 GATATACCTGTGGACTGG 607427 kkeekeekeekeekeeke 0.30 316 55754 150008 TAACGATATACCTGTGGA 607428 kkeekeekeekeekeeke 0.19 320 55758 350012 CGGTTAACGATATACCTG 607429 kkeekeekeekeekeeke 0.19 324 55762 350016 GGTGCGGTTAACGATATA 607430 kkeekeekeekeekeeke 0.41 328 n/a n/a50020 GGTGGGTGCGGTTAACGA 607431 kkeekeekeekeekeeke 0.44 416

TABLE 20EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in b.END cellsmeasured with LTS01052 Mis- matches Human to the Murine SEQ Start humanStart ISIS EDA + ID Site sequence Site Sequence No Chemistry FN NO 557130 49967 TCTCCATATCATCGTGCA 606793 eeeeeeeeeeeeeeeeee 0.19 308 55466 049720 GCAAATTAATGGTAAGAG 607360 kkeekeekeekeekeeke 0.51 417 55470 049724 TTAGGCAAATTAATGGTA 607361 kkeekeekeekeekeeke 0.53 418 55474 049728 TCTGTTAGGCAAATTAAT 607362 kkeekeekeekeekeeke 0.64 180 55478 049732 AATGTCTGTTAGGCAAAT 607363 kkeekeekeekeekeeke 1.06  32 55482 049736 GATCAATGTCTGTTAGGC 607364 kkeekeekeekeekeeke 0.85 184 55486 049740 GGGCGATCAATGTCTGTT 607365 kkeekeekeekeekeeke 1.12 185 55490 049744 TTTAGGGCGATCAATGTC 607366 kkeekeekeekeekeeke 0.44 189 55494 049748 GTCCTTTAGGGCGATCAA 607367 kkeekeekeekeekeeke 0.88 193 55498 049752 GCCAGTCCTTTAGGGCGA 607368 kkeekeekeekeekeeke 1.09 419 55502 049756 GAATGCCAGTCCTTTAGG 607369 kkeekeekeekeekeeke 1.04 420 55506 049760 CAGTGAATGCCAGTCCTT 607370 kkeekeekeekeekeeke 0.97 421 55510 049764 ACATCAGTGAATGCCAGT 607371 kkeekeekeekeekeeke 0.95 422 55514 049768 ATCCACATCAGTGAATGC 607372 kkeekeekeekeekeeke 1.05 423 55518 049772 CGACATCCACATCAGTGA 607373 kkeekeekeekeekeeke 1.04 424 55522 049776 GAATCGACATCCACATCA 607374 kkeekeekeekeekeeke 0.94 425 55526 049780 GATGGAATCGACATCCAC 607375 kkeekeekeekeekeeke 1.07 199 55530 049784 TTTTGATGGAATCGACAT 607376 kkeekeekeekeekeeke 1.05 426 55534 049788 GCAATTTTGATGGAATCG 607377 kkeekeekeekeekeeke 0.89 204 55538 049792 CCAAGCAATTTTGATGGA 607378 kkeekeekeekeekeeke 0.88 208 55542 049796 TTTCCCAAGCAATTTTGA 607379 kkeekeekeekeekeeke 0.97 427 55546 049800 GGGCTTTCCCAAGCAATT 607380 kkeekeekeekeekeeke 1.09 428 55550 049804 CTGTGGGCTTTCCCAAGC 607381 kkeekeekeekeekeeke 1.05 429 55554 049808 GCCCCTGTGGGCTTTCCC 607382 kkeekeekeekeekeeke 1.26 430 55558 049812 ACTTGCCCCTGTGGGCTT 607383 kkeekeekeekeekeeke 1.26 431 55562 049816 GGAAACTTGCCCCTGTGG 607384 kkeekeekeekeekeeke 1.07 432 55566 049820 ACCTGGAAACTTGCCCCT 607385 kkeekeekeekeekeeke 0.91 433 55570 049824 CTGTACCTGGAAACTTGC 607386 kkeekeekeekeekeeke 0.85 434 55574 049828 CACCCTGTACCTGGAAAC 607387 kkeekeekeekeekeeke 1.03 435

TABLE 21EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in b.END cellsmeasured with LTS01050 Mis- matches Human to the Murine SEQ Start humanStart ISIS EDA + ID Site sequence Site Sequence No Chemistry FN NO 557130 49967 TCTCCATATCATCGTGCA 606793 eeeeeeeeeeeeeeeeee 0.15 308 55578 049832 AGGTCACCCTGTACCTGG 607316 kkddkddkddkddkddkk 0.47 212 55582 049836 GAGTAGGTCACCCTGTAC 607317 kkddkddkddkddkddkk 0.66 216 55586 049840 GCTCGAGTAGGTCACCCT 607318 kkddkddkddkddkddkk 0.78 220 55590 049844 CAGGGCTCGAGTAGGTCA 607319 kkddkddkddkddkddkk 0.78 224 55594 049848 TCCTCAGGGCTCGAGTAG 607320 kkddkddkddkddkddkk 0.60 228 55598 049852 TCCATCCTCAGGGCTCGA 607321 kkddkddkddkddkddkk 0.72 232 55602 049856 GGATTCCATCCTCAGGGC 607322 kkddkddkddkddkddkk 0.68 236 55606 249860 TCCCGGATTCCATCCTCA 607323 kkddkddkddkddkddkk 0.69 240 55610 349864 AAGCTCCCGGATTCCATC 607324 kkddkddkddkddkddkk 0.31 244 55614 349868 GGAAAAGCTCCCGGATTC 607325 kkddkddkddkddkddkk 0.58 248 55618 349872 GCAGGGAAAAGCTCCCGG 607326 kkddkddkddkddkddkk 0.52 252 55622 149876 AGGTGCAGGGAAAAGCTC 607327 kkddkddkddkddkddkk 0.28 256 55626 149880 CATCAGGTGCAGGGAAAA 607328 kkddkddkddkddkddkk 0.45 260 55630 049884 TCACCATCAGGTGCAGGG 607329 kkddkddkddkddkddkk 0.34 264 55634 149888 GTCTTCACCATCAGGTGC 607330 kkddkddkddkddkddkk 0.16 268 55638 149892 TGTCGTCTTCACCATCAG 607331 kkddkddkddkddkddkk 0.23 272 55642 149896 GCAGTGTCGTCTTCACCA 607332 kkddkddkddkddkddkk 0.18 276 55646 149900 CTCTGCAGTGTCGTCTTC 607333 kkddkddkddkddkddkk 0.17 280 55650 149904 GCAGCTCTGCAGTGTCGT 607334 kkddkddkddkddkddkk 0.41 284 55654 149908 CCCTGCAGCTCTGCAGTG 607335 kkddkddkddkddkddkk 0.44 288 55658 149912 GAGGCCCTGCAGCTCTGC 607336 kkddkddkddkddkddkk 0.42 292 55662 249916 GCCTGAGGCCCTGCAGCT 607337 kkddkddkddkddkddkk 0.23 403 55666 249920 CCCGGCCTGAGGCCCTGC 607338 kkddkddkddkddkddkk 0.14 404 55670 249924 AGACCCCGGCCTGAGGCC 607339 kkddkddkddkddkddkk 0.15 405 55674 249928 ACTCAGACCCCGGCCTGA 607340 kkddkddkddkddkddkk 0.15 406 55678 249932 GTGTACTCAGACCCCGGC 607341 kkddkddkddkddkddkk 0.22 407 55682 149936 GACTGTGTACTCAGACCC 607342 kkddkddkddkddkddkk 0.17 408 55686 049940 CACTGACTGTGTACTCAG 607343 kkddkddkddkddkddkk 0.39 409 55690 049944 ACCACACTGACTGTGTAC 607344 kkddkddkddkddkddkk 0.58 410 55694 049948 GGCAACCACACTGACTGT 607345 kkddkddkddkddkddkk 0.38 411 55698 049952 GCAAGGCAACCACACTGA 607346 kkddkddkddkddkddkk 0.43 296 55702 049956 TCGTGCAAGGCAACCACA 607347 kkddkddkddkddkddkk 0.56 300 55706 049960 ATCATCGTGCAAGGCAAC 607348 kkddkddkddkddkddkk 0.20 412 55710 049964 CCATATCATCGTGCAAGG 607349 kkddkddkddkddkddkk 0.22 305 55714 049968 CTCTCCATATCATCGTGC 607350 kkddkddkddkddkddkk 0.44 309 55718 049972 CTGGCTCTCCATATCATC 607351 kkddkddkddkddkddkk 0.29 313 55738 149992 GACTGGATTCCAATCAGG 607352 kkddkddkddkddkddkk 0.15 413 55742 149996 TGTGGACTGGATTCCAAT 607353 kkddkddkddkddkddkk 0.51 414 55746 150000 TACCTGTGGACTGGATTC 607354 kkddkddkddkddkddkk 0.23 415 55750 150004 GATATACCTGTGGACTGG 607355 kkddkddkddkddkddkk 0.24 316 55754 150008 TAACGATATACCTGTGGA 607356 kkddkddkddkddkddkk 0.21 320 55758 350012 CGGTTAACGATATACCTG 607357 kkddkddkddkddkddkk 0.14 324 55762 350016 GGTGCGGTTAACGATATA 607358 kkddkddkddkddkddkk 0.23 328 n/a n/a50020 GGTGGGTGCGGTTAACGA 607359 kkddkddkddkddkddkk 0.25 416

TABLE 22EDA ⁺FN mRNA levels compared to untreated cells (designated 1.00) in b.END cellsmeasured with LTS01052 Mis- matches Human to the Murine SEQ Start humanStart ISIS EDA + ID Site sequence Site Sequence No Chemistry FN NO 557130 49967 TCTCCATATCATCGTGCA 606793 eeeeeeeeeeeeeeeeee 0.14 308 55466 049720 GCAAATTAATGGTAAGAG 607288 kkddkddkddkddkddkk 0.75 417 55470 049724 TTAGGCAAATTAATGGTA 607289 kkddkddkddkddkddkk 0.62 418 55474 049728 TCTGTTAGGCAAATTAAT 607290 kkddkddkddkddkddkk 0.47 180 55478 049732 AATGTCTGTTAGGCAAAT 607291 kkddkddkddkddkddkk 0.88  32 55482 049736 GATCAATGTCTGTTAGGC 607292 kkddkddkddkddkddkk 0.4 184 55486 0 49740GGGCGATCAATGTCTGTT 607293 kkddkddkddkddkddkk 0.49 185 55490 0 49744TTTAGGGCGATCAATGTC 607294 kkddkddkddkddkddkk 0.35 189 55494 0 49748GTCCTTTAGGGCGATCAA 607295 kkddkddkddkddkddkk 0.53 193 55498 0 49752GCCAGTCCTTTAGGGCGA 607296 kkddkddkddkddkddkk 0.66 419 55502 0 49756GAATGCCAGTCCTTTAGG 607297 kkddkddkddkddkddkk 0.58 420 55506 0 49760CAGTGAATGCCAGTCCTT 607298 kkddkddkddkddkddkk 0.67 421 55510 0 49764ACATCAGTGAATGCCAGT 607299 kkddkddkddkddkddkk 0.67 422 55514 0 49768ATCCACATCAGTGAATGC 607300 kkddkddkddkddkddkk 0.78 423 55518 0 49772CGACATCCACATCAGTGA 607301 kkddkddkddkddkddkk 0.72 424 55522 0 49776GAATCGACATCCACATCA 607302 kkddkddkddkddkddkk 0.65 425 55526 0 49780GATGGAATCGACATCCAC 607303 kkddkddkddkddkddkk 0.84 199 55530 0 49784TTTTGATGGAATCGACAT 607304 kkddkddkddkddkddkk 0.88 426 55534 0 49788GCAATTTTGATGGAATCG 607305 kkddkddkddkddkddkk 0.62 204 55538 0 49792CCAAGCAATTTTGATGGA 607306 kkddkddkddkddkddkk 0.78 208 55542 0 49796TTTCCCAAGCAATTTTGA 607307 kkddkddkddkddkddkk 0.59 427 55546 0 49800GGGCTTTCCCAAGCAATT 607308 kkddkddkddkddkddkk 0.61 428 55550 0 49804CTGTGGGCTTTCCCAAGC 607309 kkddkddkddkddkddkk 1.08 428 55554 0 49808GCCCCTGTGGGCTTTCCC 607310 kkddkddkddkddkddkk 1.13 430 55558 0 49812ACTTGCCCCTGTGGGCTT 607311 kkddkddkddkddkddkk 1.14 431 55562 0 49816GGAAACTTGCCCCTGTGG 607312 kkddkddkddkddkddkk 0.94 432 55566 0 49820ACCTGGAAACTTGCCCCT 607313 kkddkddkddkddkddkk 0.65 433 55570 0 49824CTGTACCTGGAAACTTGC 607314 kkddkddkddkddkddkk 0.68 434 55574 0 49828CACCCTGTACCTGGAAAC 607315 kkddkddkddkddkddkk 0.77 435

Example 11: Dose-Dependent Antisense Inhibition of Fibronectin withDeoxy, MOE and (S)-cEt Antisense Oligonucleotides in b.END Cells

Antisense oligonucleotides from the studies described above exhibitingsignificant in vitro inhibition of EDA⁺FN mRNA were selected and testedat various doses in b.END cells. Cells were transfected using Cytofectinreagent with 0.19 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.125 nM, or 6.25 nMconcentrations of antisense oligonucleotide, as specified in Tables23-27. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and EDA⁺FN mRNA levels were measured byquantitative real-time PCR. Primer probe sets LTS01050 and LTS01052 wereused to measure mRNA levels. EDA⁺FN mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN®. The results demonstrateblocking of splicing, as represented by EDA⁺FN expression. Theexpression value of untreated cells was taken as 1.00. Different primerprobe sets were used for different antisense oligonucleotide-treatedcells to avoid the amplicon effect. Each table represents a separateexperiment.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Tables 23-27. As illustrated in the tables, EDA⁺FNmRNA levels were significantly reduced in a dose-dependent manner inantisense oligonucleotide treated cells.

TABLE 23 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by 16-mer MOE, deoxy and (S)-cEt antisense oligonucleotides inb.END cells measured with LTS01050 0.19 0.39 0.78 1.56 3.125 6.25 ISISNo nM nM nM nM nM nM IC₅₀ 607178 0.7 0.6 0.5 0.3 0.2 0.1 0.59 607186 0.60.5 0.4 0.2 0.2 0.1 0.39 607188 0.8 0.6 0.4 0.3 0.2 0.1 0.67 607204 0.60.4 0.3 0.2 0.1 0.1 0.27 607205 0.6 0.5 0.4 0.3 0.2 0.1 0.34 607207 0.50.4 0.3 0.2 0.1 0.1 0.21 607208 0.9 0.5 0.4 0.3 0.2 0.2 0.62 607209 0.70.6 0.5 0.3 0.3 0.2 0.68 607210 0.8 0.6 0.5 0.3 0.3 0.2 0.77

TABLE 24 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by 16-mer MOE, deoxy and (S)-cEt antisense oligonucleotides inb.END cells measured with LTS01052 0.19 0.39 0.78 1.56 3.125 6.25 ISISNo nM nM nM nM nM nM IC₅₀ 607148 0.6 0.5 0.6 0.4 0.2 0.1 0.60 607149 0.70.5 0.3 0.3 0.1 0.1 0.44

TABLE 25 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by 18-mer MOE, deoxy and (S)-cEt antisense oligonucleotides inb.END cells measured with LTS01050 0.19 0.39 0.78 1.56 3.125 6.25 ISISNo nM nM nM nM nM nM IC₅₀ 607330 0.6 0.4 0.3 0.2 0.2 0.1 0.28 607332 0.60.5 0.3 0.2 0.1 0.1 0.35 607333 0.6 0.6 0.5 0.3 0.2 0.2 0.59 607338 0.80.9 0.8 0.7 0.5 0.4 3.64 607339 0.8 0.8 0.7 0.6 0.6 0.5 5.89 607340 0.70.6 0.5 0.3 0.3 0.2 0.63 607341 0.6 0.5 0.4 0.3 0.2 0.1 0.44 607342 0.90.7 0.5 0.5 0.4 0.4 1.54 607348 0.9 0.7 0.5 0.3 0.2 0.2 0.96 607352 0.40.4 0.2 0.1 0.1 0.1 0.16 607357 0.7 0.5 0.4 0.2 0.2 0.1 0.48

TABLE 26 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by 16-mer MOE, deoxy and (S)-cEt antisense oligonucleotides inb.END cells measured with LTS01050 0.19 0.39 0.78 1.56 3.125 6.25 ISISNo nM nM nM nM nM nM IC₅₀ 607245 0.9 0.9 0.5 0.5 0.4 0.4 2.04 607275 0.80.8 0.5 0.5 0.2 0.2 1.09 607276 0.8 0.6 0.4 0.3 0.2 0.2 0.57 607277 0.70.6 0.4 0.2 0.2 0.1 0.48 607284 0.6 0.6 0.3 0.1 0.1 0.1 0.37 607285 0.40.4 0.2 0.1 0.1 0.1 0.12 607286 0.7 0.4 0.3 0.2 0.1 0.1 0.36

TABLE 27 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by MOE, deoxy and (S)-cEt antisense oligonucleotides in b.ENDcells measured with LTS01050 Length 0.19 0.39 0.78 1.56 3.125 6.25 ISISNo (nt) nM nM nM nM nM nM IC₅₀ 607428 18 0.5 0.3 0.1 0.1 0.1 0.1 0.16607429 18 0.6 0.7 0.5 0.3 0.2 0.1 0.58 607213 16 0.9 0.8 0.5 0.3 0.2 0.20.95

Example 12: Dose-Dependent Antisense Inhibition of Fibronectin withUniform MOE Antisense Oligonucleotides in b.END Cells

Antisense oligonucleotides from the studies described above exhibitingsignificant in vitro inhibition of EDA⁺FN mRNA were selected and testedat various doses in b.END cells. Cells were transfected using Cytofectinreagent with 0.39 nM, 0.78 nM, 1.56 nM, 3.125 nM, 6.25 nM or 12.5 nMconcentrations of antisense oligonucleotide, as specified in Tables28-31. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and EDA⁺FN mRNA levels were measured byquantitative real-time PCR. Primer probe sets LTS01050 and LTS01052 wereused to measure mRNA levels. EDA⁺FN mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN®. The results demonstrateblocking of splicing, as represented by EDA⁺FN expression. Theexpression value of untreated cells was taken as 1.00. Different primerprobe sets were used for different antisense oligonucleotide-treatedcells to avoid the amplicon effect. Each table represents a separateexperiment.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Tables 28-31. As illustrated in the tables, EDA⁺FNmRNA levels were significantly reduced in a dose-dependent manner inantisense oligonucleotide treated cells.

TABLE 28 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by 18-mer uniform MOE oligonucleotides in b.END cells measuredwith LTS01050 0.39 0.78 1.56 3.125 6.25 12.5 ISIS No nM nM nM nM nM nMIC₅₀ 606708 1.1 0.8 0.6 0.4 0.3 0.2 2.79 606723 0.8 0.8 0.6 0.4 0.2 0.12.39 606729 0.8 0.6 0.3 0.2 0.1 0.1 1.10 606753 0.8 0.5 0.4 0.3 0.2 0.11.09 606770 0.7 0.5 0.4 0.2 0.1 0.1 0.87 606785 0.5 0.3 0.2 0.1 0.1 0.10.37 606787 0.5 0.3 0.3 0.1 0.1 0.1 0.30 606788 0.4 0.2 0.2 0.1 0.1 0.10.21 606793 0.3 0.3 0.2 0.1 0.1 0.1 0.09 606804 0.5 0.3 0.3 0.1 0.1 0.10.40 606812 0.4 0.3 0.2 0.1 0.1 0.1 0.23

TABLE 29 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by ISIS 606675 in b.END cells measured with LTS01052 0.39 nM 0.80.78 nM 0.6 1.56 nM 0.5 3.125 nM  0.3 6.25 nM 0.1 12.5 nM 0.1 IC₅₀ 1.38

TABLE 30 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by 15-mer uniform MOE oligonucleotides in b.END cells measuredwith LTS01050 0.39 0.78 1.56 3.125 6.25 12.5 ISIS No nM nM nM nM nM nMIC₅₀ 598137 1.0 0.8 0.7 0.5 0.4 0.2 4.08 598138 0.8 0.9 0.6 0.6 0.4 0.24.11 598144 0.9 0.7 0.4 0.4 0.2 0.1 1.75 598151 0.7 0.5 0.3 0.2 0.1 0.10.79 598153 0.9 0.6 0.5 0.3 0.2 0.1 1.54 598161 0.9 0.5 0.5 0.3 0.2 0.21.50 598163 0.6 0.5 0.4 0.3 0.1 0.1 0.60 594675 0.8 0.6 0.4 0.3 0.1 0.11.18 598145 0.9 0.7 0.5 0.3 0.2 0.1 1.58

TABLE 31 EDA⁺FN mRNA levels compared to untreated cells (designated1.00) by 15-mer uniform MOE oligonucleotides in b.END cells measuredwith LTS01052 0.39 0.78 1.56 3.125 6.25 12.5 ISIS No nM nM nM nM nM nMIC₅₀ 511404 0.9 0.8 0.7 0.4 0.3 0.2 2.81 598130 0.9 1.0 0.9 0.9 0.7 0.513.18

Example 13: Efficacy and Tolerability of Antisense OligonucleotidesTargeting Fibronectin in C57BL/6 Mice

C57BL/6 mice are a multipurpose mice model, frequently utilized forsafety and efficacy testing. The mice were treated with ISIS antisenseoligonucleotides selected from studies described above and evaluated forefficacy, as well as changes in the levels of various plasma chemistrymarkers.

Study with Uniform MOE Oligonucleotides

Treatment

Groups of eight-week old C57BL/6 mice were injected subcutaneously twicea week for 3 weeks with 100 mg/kg of ISIS 594675, ISIS 598145, ISIS598151, ISIS 598153, ISIS 598163, ISIS 606770, ISIS 606785, ISIS 606787,ISIS 606788, ISIS 606793, ISIS 606804, or ISIS 606812. One group ofeight-week old C57BL/6 mice was injected subcutaneously twice a week for3 weeks with PBS. Mice were euthanized 48 hours after the last dose, andorgans and plasma were harvested for further analysis.

RNA Analysis

To evaluate the effect of ISIS oligonucleotides on blocking fibronectinsplicing, mRNA levels of EDA⁺FN were measured by RT-PCR using mouseprimer probe set LTS01050 and LTS01052. The results are presented inTable 32, normalized to total fibronectin. The results demonstrateblocking of splicing, as represented by EDA⁺FN expression. Theexpression value in untreated mice was taken as 1.00. ‘n.d.’ indicatesthat the mRNA expression level values were not considered because theoligonucleotide targeted an amplicon region of the specific primer probeset.

TABLE 32 EDA⁺FN expression after antisense oligonucleotide treatment inC57BL/6 mice Lungs Kidneys EDA⁺FN/ EDA⁺FN/ EDA⁺FN/ EDA⁺FN/ total FNtotal FN total FN total FN ISIS No (LTS01050) (LTS01052) (LTS01050)(LTS01052) 594675 0.43 0.43 0.52 0.57 598145 0.65 0.61 0.61 0.64 5981510.40 0.33 0.43 0.36 598153 0.75 0.57 1.07 0.69 598163 0.24 0.23 0.150.17 606770 0.50 0.38 0.54 0.52 606785 0.31 0.33 0.22 0.29 606787 0.360.32 0.26 0.32 606788 0.40 0.36 0.19 0.22 606793 0.28 0.23 0.12 0.14606804 0.32 n.d. 0.27 n.d. 606812 0.39 n.d. 0.42 n.d.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.). The results are presented in Table 33.ISIS oligonucleotides did not cause any changes in the levels of any ofthe liver or kidney function markers outside the expected range forantisense oligonucleotides.

TABLE 33 Plasma chemistry markers in C57BL/6 mice plasma ALT ASTBilirubin BUN (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 26 46 0.19 35 ISIS594675 19 42 0.20 31 ISIS 598145 25 49 0.20 29 ISIS 598151 30 67 0.18 35ISIS 598153 26 71 0.17 34 ISIS 598163 64 102 0.19 38 ISIS 606770 22 540.18 29 ISIS 606785 48 94 0.17 32 ISIS 606787 30 71 0.15 30 ISIS 60678882 116 0.15 33 ISIS 606793 50 83 0.15 32 ISIS 606804 31 56 0.16 28 ISIS606812 29 49 0.15 27

Organ Weights

Liver, spleen and kidney weights were measured at the end of the study,and are presented in Table 34. ISIS oligonucleotides did not cause anychanges in organ weights outside the expected range for antisenseoligonucleotides.

TABLE 34 Organ weights (% of the PBS control) of C57BL/6 mice ISIS NoLiver Kidneys Spleen 594675 102 102 99 598145 98 105 117 598151 108 10390 598153 106 100 88 598163 102 102 94 606770 106 104 93 606785 101 10298 606787 108 99 93 606788 98 97 99 606793 103 94 99 606804 95 98 89606812 105 101 86Study with Deoxy, (S)-cEt and MOE Oligonucleotides

Treatment

Groups of eight-week old C57BL/6 mice were injected subcutaneously twicea week for 3 weeks with 100 mg/kg of ISIS 607149, ISIS 607186, ISIS607204, ISIS 607205, ISIS 607207, ISIS 607277, ISIS 607285, ISIS 607286,ISIS 607330, ISIS 607332, ISIS 607341, ISIS 607352, ISIS 607428, or ISIS607429. One group of eight-week old C57BL/6 mice was injectedsubcutaneously twice a week for 3 weeks with PBS. Mice were euthanized48 hours after the last dose, and organs and plasma were harvested forfurther analysis.

RNA Analysis

To evaluate the effect of ISIS oligonucleotides on blocking fibronectinsplicing, mRNA levels of EDA⁺FN were measured by RT-PCR using mouseprimer probe set LTS01050 and LTS01052. The results are presented inTable 35, normalized to total fibronectin. The results demonstrateblocking of splicing, as represented by EDA⁺FN expression. Theexpression value in untreated mice was taken as 1.00. ‘n.d.’ indicatesthat the mRNA expression level values were not considered because theoligonucleotide targeted an amplicon region of the specific primer probeset.

TABLE 35 EDA⁺FN expression after antisense oligonucleotide treatment inC57BL/6 mice Lungs Kidneys EDA⁺FN/ EDA⁺FN/ EDA⁺FN/ EDA⁺FN/ total FNtotal FN total FN total FN ISIS No (LTS01050) (LTS01052) (LTS01050)(LTS01052) 607149 n.d. 0.32 n.d. 0.07 607186 0.26 0.14 0.02 0.02 6072040.15 0.13 0.02 0.03 607205 0.30 n.d. 0.03 n.d. 607207 0.42 n.d. 0.04n.d. 607277 0.33 0.27 0.05 0.08 607285 0.21 n.d. 0.29 n.d. 607286 0.13n.d. 0.02 n.d. 607330 0.29 0.27 0.02 0.06 607332 0.08 0.13 0.01 0.05607341 0.15 0.24 0.10 0.16 607352 0.11 n.d. 0.01 n.d. 607357 0.12 n.d.0.09 n.d. 607428 0.12 n.d. 0.07 n.d. 607429 0.33 n.d. 0.20 n.d.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.). The results are presented in Table 36.ISIS oligonucleotides that caused any changes in organ weights outsidethe expected range for antisense oligonucleotides were excluded fromfurther studies.

TABLE 36 Plasma chemistry markers in C57BL/6 mice plasma ALT ASTBilirubin BUN (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 38 96 0.18 36 ISIS607149 35 170 0.29 31 ISIS 607186 22 73 0.21 26 ISIS 607204 41 75 0.1529 ISIS 607205 38 83 0.15 32 ISIS 607207 70 92 0.19 32 ISIS 607277 54108 0.16 30 ISIS 607285 78 139 0.32 28 ISIS 607286 40 94 0.38 27 ISIS607330 23 40 0.16 30 ISIS 607332 41 66 0.20 28 ISIS 607341 70 102 0.2428 ISIS 607352 20 75 0.13 27 ISIS 607357 85 100 0.15 26 ISIS 607428 2182 0.24 25 ISIS 607429 21 50 0.17 26

Organ Weights

Liver, spleen and kidney weights were measured at the end of the study,and are presented in Table 37. ISIS oligonucleotides that caused anychanges in organ weights outside the expected range for antisenseoligonucleotides were excluded from further studies.

TABLE 37 Organ weights (% of the PBS control) of C57BL/6 mice ISIS No.Liver Kidneys Spleen 607149 103 103 99 607186 91 106 101 607204 93 97116 607205 112 114 276 607207 98 99 96 607277 98 100 108 607285 99 97105 607286 96 103 98 607330 97 101 85 607332 102 98 97 607341 93 98 92607352 98 101 84 607357 88 101 113 607428 96 101 90 607429 91 101 99

1-118. (canceled)
 119. A compound comprising a modified oligonucleotideconsisting of 12 to 30 linked nucleosides and having a nucleobasesequence comprising a complementary region comprising at least 12contiguous nucleobases complementary to a target region of equal lengthof a fibronectin transcript, wherein the target region is withinnucleobase 55469 and nucleobase 55790 of SEQ ID NO.:
 1. 120. Thecompound of claim 119, wherein the complementary region of the modifiedoligonucleotide comprises at least 15 contiguous nucleobases.
 121. Thecompound of claim 119, wherein the complementary region of the modifiedoligonucleotide comprises at least 18 contiguous nucleobases.
 122. Thecompound of claim 119, wherein the complementary region of the modifiedoligonucleotide comprises at least 20 contiguous nucleobases.
 123. Thecompound of claim 119, wherein the complementary region of the modifiedoligonucleotide is 100% complementary to the target region.
 124. Thecompound of claim 120, wherein the complementary region of the modifiedoligonucleotide is 100% complementary to the target region.
 125. Thecompound of claim 121, wherein the nucleobase sequence of theoligonucleotide is at least 90% complementary to an equal-length regionof the fibronectin transcript, as measured over the entire length of theoligonucleotide.
 126. The compound of claim 119, wherein the modifiedoligonucleotide comprises at least one modified nucleoside comprising amodified sugar moiety.
 127. The compound of claim 126, wherein themodified sugar moiety is a 2′-substituted sugar moiety, wherein the2′-substituent is selected from among: 2′-OMe, 2′-F, and 2′-MOE. 128.The compound of claim 126, wherein the modified sugar moiety is abicyclic sugar moiety.
 129. The compound of claim 128, wherein thebicyclic sugar moiety is LNA or cEt.
 130. The compound of claim 126,wherein the modified sugar moiety is a sugar surrogate, wherein thesugar surrogate is a morpholino or a modified morpholino.
 131. Thecompound of claim 126, wherein the modified oligonucleotide comprises atleast two modified nucleosides comprising modified sugar moieties thatare different from one another.
 132. The compound of claim 126, whereinthe modified oligonucleotide comprises at least two modified nucleosidesthat have the same 2′-substituted sugar moiety.
 133. The compound ofclaim 132, wherein the 2′-substituent of the 2′-substituted sugar moietyis 2′-MOE.
 134. The compound of claim 119, wherein the modifiedoligonucleotide comprises at least one modified internucleoside linkage.135. The compound of claim 134, comprising at least one phosphorothioateinternucleoside linkage.
 136. A pharmaceutical composition comprising acompound according to claim 119 and a pharmaceutically acceptablecarrier or diluent.
 137. The pharmaceutical composition of claim 136,wherein the pharmaceutically acceptable carrier or diluent is sterilesaline.